Pancreas-derived plasminogen activator inhibitor

ABSTRACT

The present invention relates to a novel member of the plasminogen activator inhibitor protein family. In particular, isolated nucleic acid molecules are provided encoding the pancreas-derived plasminogen activator inhibitor protein. Pancreas-derived plasminogen activator inhibitor polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to methods for treating physiologic and pathologic disease conditions, including breast cancer, and diagnostic methods for detecting pathologic disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/934,011, filed Aug. 15, 1997, which is herein incorporated byreference; said U.S. application Ser. No. 08/934,011 claims prioritybenefit to U.S. application Ser. No. 60/024,056, filed Aug. 16, 1996,which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel member of the serine proteaseinhibitor (serpin) superfamily of proteins, in particular theplasminogen activator inhibitor (PAI) protein family. More specifically,isolated nucleic acid molecules are provided encoding thepancreas-derived plasminogen activator inhibitor (PAPAI) protein.Plasminogen activator inhibitor polypeptides are also provided. Thepresent invention further relates to methods for treating physiologicand pathologic disease conditions and diagnostic methods for detectingpathologic disorders.

2. Related Art

The mammalian serine protease inhibitors (serpins) are a superfamily ofsingle chain proteins that contain a conserved structure ofapproximately 370 amino acids and generally range between 40 and 60 kDain molecular mass. α₁-Antitrypsin (also known as α₁-proteinaseinhibitor) is a characteristic member of the serpin family in that it isa single chain glycoprotein of nearly 400 amino acid residues thatfunctions by forming a tight 1:1 complex with its cognate protease,neutrophil (leucocyte) elastase, which subsequently slowly dissociatesto yield active enzyme and inactive cleaved inhibitor (Carrell, R. W. etal., Cold Spring Harbor Symposia on Quantitative Biology 52:527-535(1987)). The reactive center of the serpins is typically formed by anX-Ser that acts as a substrate for the target serine protease:α₁-antitrypsin has a Met-Ser reactive center with the methionine residueproviding a putative cleavage site for neutrophil elastase.

The majority of serpins function as protease inhibitors and so areinvolved in regulation of several proteinase-activated physiologicalprocesses, such as blood coagulation, fibrinolysis, complementactivation, extracellular matrix turnover, cell migration and prohormoneactivation (Potempa, J. et al., J. Biol. Chem. 269:15957-19560 (1994)).As noted, serpins inhibit proteolytic events by forming a 1:1stoichiometric complex with the active site of their cognateproteinases, which is resistant to denaturants (Cohen, A. B. et al.,Biochemistry 17:392-400 (1987). The serpins include, but are not limitedto, α₁-antitrypsin (α₁-proteinase inhibitor), antithrombin III,plasminogen activator inhibitor 1 (PAI-1), plasminogen activatorinhibitor 2 (PAI-2), α₁-antichymotrypsin, and α₂-antiplasmin (Huber, R.and Carrell, R. W., Biochemistry 28:8951-8966 (1989).

The plasminogen activator system is responsible for the degradation ofintravascular blood clots, and also contributes to extracellularproteolysis in a wide variety of physiological processes of normaldevelopment and pathological processes in the etiology of diseases suchas tumor invasion and metastasis (Andreasen, P.-A., et al., Int. J.Cancer 72(1):1-22 (1997); Schmitt, M., et al., Thromb. Haemost.78(1):285-296 (1997)). Plasmin, a trypsin-like protease, is generatedfrom its precursor plasminogen by the action of plasminogen activators,of which there are two types: tissue-type plasminogen activator (alsoknown as tissue plasminogen activator) and urokinase. Plasmin degradesfibrin and several extracellular matrix and adhesion proteins andactivates procollagenases.

Plasminogen activation is a highly regulated process. Precise,coordinated, spatial and temporal regulation is afforded by theinteraction of a variety of mechanisms. These mechanisms include (1)inhibition by specific plasmin and plasminogen activator inhibitors; (2)binding of plasminogen, plasminogen activators, and inhibitors tofibrin, extracellular matrix proteins, and specific cell surfacereceptors; (3) release of tissue plasminogen activator and inhibitorsfrom intracellular storage granules; (4) regulation of gene expressionof plasminogen activators and inhibitors; (5) an autocrine feedback loopwhereby plasmin-mediated activation of latent forms of growth factorsregulates the expression of activators and inhibitors; and (6) clearanceof free and inhibitor-bound activators via receptors (Bachmann, F. etal., Fibrinolysis, in: Thrombosis Haemostasis 1987, Verstraete, M. etal., eds., Leuven University Press (1987); Danø, K. et al., Adv. CancerRes. 44:139 (1985); Pöllänen, J. et al., Adv. Cancer Res. 57:273 (1991);Vassalli, J. D. et al., J. Clin. Invest. 88:1067 (1991); Carmeliet, P.et al., Thromb. Haemost. 74:429 (1995); Andreasen, P. A. et al., Mol.Cell. Endocrinol. 68:1 (1990); Loskutoff, D. J., Fibrinolysis 5:197(1991); Keski-Oja, J. et al., Semin. Thromb. Hemost. 17:231 (1991);Blasi, F., BioEssays 15:105 (1993); Andreasen, P. A. et al., FEBS Lett.338:239 (1994); Bu, G. et al., Blood 83:3427 (1994); and Camani, C. etal., Int. J. Hematol. 60:97 (1994)).

Strong clinical and experimental evidences have suggested a causal rolefor the tumor-associated urokinase-type PA (u-PA) and the receptor u-PARin cancer invasion and metastasis (Andreasen, P.-A., et al., Int. J.Cancer 72(1):1-22 (1997); Schmitt, M., et al., Thromb. Haemost.78(1):285-296 (1997); Duggan, C., et al., Br. J. Cancer 76(5):622-627(1997)). Consistent with its role in cancer metastasis, overexpressionand unrestrained activity of u-PA has been shown to be a prognosticmarker in many different types of human cancer (Schmitt, M., et al.,Fibrinolysis 6(Suppl. 4):3-26 (1992); Schmitt, M., et al., J. Obstet.Gynaecol. 21:151-165 (1995); Brunner, N., et al., Cancer Treat. Res.71:299-309 (1994); Kuhn, W., et al., Gynecol. Oncol. 55:401-409 (1994);Ganesh, S., et al., Cancer Res. 54:4065-4071 (1994); Nekarda, H., etal., Cancer Res. 54:2900-2907 (1994); Duffy, M.-J., J. Clin. Cancer Res.2:613-618 (1996)). The down-regulation of u-PA may occur at the levelsof transcriptional regulation of the genes and through interaction withspecific endogenous inhibitors such as plasminogen activator inhibitor(PAI).

Only two plasminogen activator inhibitors are known. These areplasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2, respectively).PAI-1 and PAI-2 regulate mitogenesis, adhesion of myeloid cells, fusionof myoblasts, and migration of endothelial cells (Fazioli, F. et al.,Trends Pharm. Sci. 15:25-29 (1995)). Indeed, PAI-1 and PAI-2 areinvolved in many physiological and pathological processes, includingnormal pregnancy, preeclampsia, intrauterine growth retardation, woundhealing, tumor cell invasion and metastasis, inflammation and arthritis,inflammatory bowel disease, appendicitis, complications from systemiclupus erythematosus, ovulation and prostatic involution andosteonecrosis (Kruithof, E. K. O. et al., Blood 86:4007 (1995)).

Both PAI-1 and PAI-2 have been shown to inhibit extracellular matrixdegradation in vitro (Cajot, J.-F., et al., Proc. Natl. Acad Sci. USA87:6939-6943 (1990); Baker, M.-S., et al., Cancer Res. 50:4676-4684(1990)). These results suggest that the inhibitory activity of PAlsmight be important in inhibiting tumor malignant progression leading tometastasis. In fact, administration of a recombinant PAI-2 to micedecreases tumor growth (Astedt, B., et al, Fibrinol. 9:175-177 (1995)),whereas overexpression of either PAI-1 or PAI-2 inhibits tumormetastasis (Muller, B., et al., Proc. Natl. Acad Sci. USA 92:205-209(1995); Soff, G.-A., et al., J. Clin. Invest. 96:2593-2600 (1995)).

In breast cancer, it has been reported that uPA and PAI-1 arestatistically independent, strong prognostic factors for disease freeand overall survival, i.e., high tumor levels are associated with a poorprognosis and are conductive to tumor cell spread and metastasis(Brunner, N., et al., Cancer Treat. Res. 71:299-309 (1994); Duffy, M.,et al., Cancer 62:531-533 (1988); Duggan, C., et al., Int. J. Cancer61:597-600 (1995); Schmitt, M., et al., Br. J. Cancer 76(3):306-311(1997)). Immunohistochemical staining has detected PAI-I expression atstromal fibroblasts surrounding tumor nodules or at tumor margins(Bianchi, E., et al., Int. J. Cancer 60:597-603 (1995)). The productionof PAI-I by the tumor stroma may represent a host defensive response tothe excessive proteolysis. In contrast to PAI-I, high level PAI-2expression may be a favorable prognostic marker in breast cancer(Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997); Duggan, C.,et al., Br. J. Cancer 76(5):622-627 (1997)). In breast carcinomas withhigh uPA values, PAI-2 was associated with a prolonged relapse-freesurvival, metastasis-free survival, and overall survival (Bouchet, C.,et al., Br. J Cancer 69:398-405 (1994)). In relation to theclinicopathological findings, an inverse correlation between PAI-2 mRNAexpression and lymph node metastasis was reported in breast cancers(Sumiyoshi, K., et al., S. Int. J. Cancer 50:345-348 (1992)). In thisstudy, the expression of uPA and PAI-1 was significantly correlated withnegative expression of PAI-2; and a low level of PAI-2 expression wassignificantly associated with lymph node involvement (Sumiyoshi, K., etal., Int. J. Cancer 50:345-348 (1992)). PAI-2 expression is detectedpredominantly in malignant mammary epithelial cells of primarycarcinomas but is also present in stromal cells (Andreasen, P.-A., etal., Int. J. Cancer 72(1):1-22 (1997)). These results indicate thatPAI-2 may play a critical role in inhibition of extracellular matrixdegradation mediated by plasminogen activator during tumor cell invasionand metastasis.

In view of the wide range of roles that plasminogen activator inhibitorsplay in physiologic and pathologic processes, there is a continuing needfor the isolation and characterization of novel plasminogen activatorinhibitors.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the pancreas-derived plasminogenactivator inhibitor (PAPAI) polypeptide having the amino acid sequenceis shown in FIG. 1 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:13) or the aminoacid sequence encoded by the cDNA clone deposited in a bacterial host asATCC Deposit Number 97657 on Jul. 12, 1996. The nucleotide sequencedetermined by sequencing the deposited PAPAI clone, which is shown inFIG. 4, contains an open reading frame encoding a polypeptide of 405amino acid residues, including an initiation codon at positions 67-69,with a leader sequence of about 18 amino acid residues, and a deducedmolecular weight of about 46 kDa. The amino acid sequence of the maturePAPAI protein is shown in SEQ ID NO:13 (amino acid residues from about 1to about 387 in SEQ ID NO:13). Another sequence of a PAPAI clone whichis shown in FIG. 1 (SEQ ID NO:1), contains an open reading frameencoding a polypeptide of 392 amino acid residues, including aninitiation codon at positions 67-69, with a leader sequence of about 14amino acid residues, and a deduced molecular weight of about 44.5 kDa.The amino acid sequence of this mature PAPAI protein is shown in SEQ IDNO:2 (amino acid residues from about 1 to about 378 in SEQ ID NO:2).

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the PAPAI polypeptide having the complete amino acid sequencein SEQ ID NO:2; (b) a nucleotide sequence encoding the PAPAI polypeptidehaving the complete amino acid sequence in SEQ ID NO:2 but minus theN-terminal methionine residue; (c) a nucleotide sequence encoding themature PAPAI polypeptide having the amino acid sequence at positions1-378 in SEQ ID NO:2; (d) a nucleotide sequence encoding the PAPAIpolypeptide having the complete amino acid sequence in SEQ ID NO:13; (e)a nucleotide sequence encoding the PAPAI polypeptide having the completeamino acid sequence in SEQ ID NO:13, but minus the N-terminal methionineresidue; (f) a nucleotide sequence encoding the mature PAPAI polypeptidehaving the amino acid sequence at positions 1-387 in SEQ ID NO:13; (g) anucleotide sequence encoding the PAPAI polypeptide having the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97657; (h) a nucleotide sequence encoding the mature PAPAIpolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97657; and (i) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), or (h) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical, to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h), or (i) above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide in (a),(b), (c), (d), (e), (f), (g), (h), or (i) above. This polynucleotidewhich hybridizes does not hybridize under stringent hybridizationconditions to a polynucleotide having a nucleotide sequence consistingof only A residues or of only T residues. An additional nucleic acidembodiment of the invention relates to an isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a PAPAI polypeptide having an amino acidsequence in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofPAPAI polypeptides or peptides by recombinant techniques.

The invention further provides an isolated PAPAI polypeptide havingamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of the PAPAI polypeptide having the complete 392 aminoacid sequence, including the leader sequence shown in SEQ ID NO:2; (b)the amino acid sequence of the PAPAI polypeptide having the complete 392amino acid sequence, including the leader sequence shown in SEQ ID NO:2,but minus the N-terminal methionine residue; (c) the amino acid sequenceof the mature PAPAI polypeptide (without the leader) having the aminoacid sequence at positions 1-378 in SEQ ID NO:2; (d) the amino acidsequence of the PAPAI polypeptide having the complete 405 amino acidsequence, including the leader sequence shown in SEQ ID NO:13; (e) theamino acid sequence of the PAPAI polypeptide having the complete 405amino acid sequence, including the leader sequence shown in SEQ IDNO:13, but minus the N-terminal methionine residue; (f) the amino acidsequence ofthe mature PAPAI polypeptide (without the leaser) having theamino acid sequence at positions 1-387 in SEQ ID NO:13; (g) the aminoacid sequence of the PAPAI polypeptide having the complete amino acidsequence, including the leader, encoded by the cDNA clone contained inATCC Deposit No. 97657; and (h) the amino acid sequence ofthe maturePAPAI polypeptide having the amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97657. The polypeptides of thepresent invention also include polypeptides having an amino acidsequence at least 95% identical, more preferably at least 96%, 97%, 98%or 99% identical to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a PAPAI polypeptide having an amino acidsequence described in (a), (b), (c), (d), (e), (f), (g), or (h) above.Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a PAPAI polypeptide of the invention includeportions of such polypeptides with at least six or seven, preferably atleast nine, and more preferably at least about 30 amino acids to about50 amino acids, although epitope-bearing polypeptides of any length upto and including the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention. In anotherembodiment, the invention provides an isolated antibody that bindsspecifically to a PAPAI polypeptide having an amino acid sequencedescribed in (a), (b), (c), (d), (e), (f), (g), or (h) above.

For a number of pathologic disorders, such as tumor invasion andmetastasis, significant alterations (increases or decreases) in level ofPAPAI gene expression can be detected in a sample of tissue or bodilyfluid. Increased or decreased levels of PAPAI gene expression can bemeasured, in such a sample, relative to a “standard” PAPAI geneexpression level, i.e., the PAPAI expression level in a tissue or bodilyfluid from an individual not having the disorder. Thus, the presentinvention provides a diagnostic method useful during diagnosis of suchdisorders, which involves assaying the expression level of the geneencoding the PAPAI protein in tissue or bodily fluid from an individualand comparing the gene expression level with a standard PAPAI geneexpression level, whereby an increase or decrease in the gene expressionlevel over the standard is indicative of a pathologic disorder, such astumor invasion and metastasis, hemorrhage in liver disease, andpreeclampsia.

The PAPAI protein inhibits the plasminogen activator system whenadministered to an individual. The plasminogen activator system isresponsible for the degradation of intravascular blood clots, while alsocontributing to extracellular proteolysis in a wide variety ofphysiological processes (e.g. wound healing, cell migration, tissueremodeling, angiogenesis, trophoblast implantation, ovulation and fetaldevelopment) and pathological processes (e.g. tumor invasion andmetastasis, intrauterine growth retardation, preeclampsia, and acute andchronic inflammation). Thus, by the invention, methods are provided forinhibiting the plasminogen activator system, which involve administeringan inhibitory amount of PAPAI either alone or together with one or moreplasminogen activator inhibitors, such as PAI-1 and PAI-2.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show the nucleotide (SEQ ID NO:1) and deduced amino acid(SEQ ID NO:2) sequences of pancreas-derived plasminogen activatorinhibitor (PAPAI) protein. The protein has a leader sequence of about 14amino acid residues (underlined) and a deduced molecular weight of about44.5 kDa. The predicted amino acid sequence of the mature PAPAI proteinis also shown in FIGS. 1A-1B (SEQ ID NO:2).

FIGS. 2A-2C show the regions of similarity between the amino acidsequences of the PAPAI protein (HPASD5OP protein) and human plasminogenactivator inhibitor 1 (PAI-1) (SEQ ID NO:3) and human plasminogenactivator inhibitor 2 (PAI-2) (SEQ ID NO:4).

FIG. 3 shows an analysis of the predicted alpha, beta, turn, and coilregions, and the predicted hydrophilicity, amphipathic nature, flexibleregions, antigenic index, and surface probability plot of the of thepolypeptide of FIG. 1 (SEQ ID NO:2) and FIG. 4 (SEQ ID NO:13). In the“Antigenic Index—Jameson-Wolf” graph, amino acid residues about 20 toabout 30, about 45 to about 50, about 60 to about 90, about 125 to about135, about 160 to about 175, about 220 to about 225, about 250 to about260, about 320 to about 330, and about 375 to about 380 in FIG. 1 andFIG. 4 correspond to the highly antigenic regions of the PAPAI protein.These highly antigenic fragments in FIG. 1 correspond to the followingfragments, respectively, in SEQ ID NO:2: amino acid residues about 6 toabout 16, about 31 to about 36, about 46 to about 76, about 111 to about121, about 146 to about 161, about 206 to about 211, about 236 to about246, about 306 to about 316, and about 361 to about 366. These highlyantigenic fragments in FIG. 4 correspond to the following fragments,respectively, in SEQ ID NO:13: amino acid residues about 2 to about 12,about 27 to about 32, about 42 to about 72, about 107 to about 117,about 142 to about 157, about 202 to about 207, about 232 to about 242,about 302 to about 312, and about 357 to about 362.

FIGS. 4A-4B show the nucleotide (SEQ ID NO:12) and deduced amino acid(SEQ ID NO:13) sequences of PAPAI, which was determined by sequencingthe cDNA clone deposited as ATCC Deposit No. 97657. The protein has aleader sequence of about 18 amino acid residues (underlined) and adeduced molecular weight of about 46 kDa.

FIG. 5 shows a schematic representation of the pHE4-5 expression vector(SEQ ID NO:14) and the subdloned PAPAI cDNA coding sequence. Thelocations of the kanamycin resistance marker gene, the PAPAI codingsequence, the oriC sequence, and the lacIq coding sequence areindicated.

FIG. 6 shows the nucleotide sequence of the regulatory elements of thepHE promoter (SEQ ID NO:15). The two lac operator sequences, theShine-Delgarno sequence (S/D), and the terminal HindIII and NdeIrestriction sites (italicized) are indicated.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a PAPAI polypeptide, having theamino acid sequence shown in FIG. 4 (SEQ ID NO:13), which was determinedby sequencing a cloned cDNA, or in FIG. 1 (SEQ ID NO:2). The PAPAIprotein of the present invention shares sequence homology with humanplasminogen activator inhibitor 1 (PAI-1) (SEQ ID NO:3) and humanplasminogen activator 2 (PAI-2)(SEQ ID NO:4). The nucleotide sequenceshown in FIG. 4 (SEQ ID NO:13) was obtained by sequencing the HPASD50Pclone, which was deposited on Jul. 12, 1996 at the American Type CultureCollection, Patent Depository, 10801 University Boulevard, Manassas Va.,20110-2209 and given accession number 97657.

Accordingly, in one embodiment of the present invention, isolatednucleic acid molecules are provided which encode the PAPAI protein.PAPAI is a novel member of the plasminogen activator inhibitorsubfamily.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence ofribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyzibonucleotide sequence is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofSEQ ID NO:1 set forth using deoxyribonucleotide abbreviations isintended to indicate an RNA molecule having a sequence in which eachdeoxyribonucleotide A, G or C of SEQ ID NO:1 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxyribonucleotide Thas been replaced by a ribonucleotide U.

Using the information provided herein, such as the nucleotide sequencein SEQ ID NO:1 or SEQ ID NO:12, a nucleic acid molecule of the presentinvention encoding a PAPAI polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative ofthe invention, the nucleicacid molecules described in SEQ ID NO:1 and SEQ ID NO:12 were discoveredin a cDNA library derived from human pancreatic tissue. The determinednucleotide sequence of the PAPAI cDNA of SEQ ID NO:1 contains an openreading frame encoding a protein of 392 amino acid residues, with aninitiation codon at positions 67-69 of the nucleotide sequence in SEQ IDNO:1, a predicted leader sequence of about 14 amino acid residues, and adeduced molecular weight of about 44.5 kDa. The amino acid sequence ofthe predicted mature PAPAI is shown in SEQ ID NO:2 from amino acidresidue 1 to residue 378. The PAPAI protein shown in SEQ ID NO:2 isabout 67% and 68% identical to PAI-1(SEQ ID NO:3) and PAI-2(SEQ IDNO:4), respectively (FIG. 2). The determined nucleotide sequence of thePAPAI cDNA of SEQ ID NO:12 contains an open reading frame encoding aprotein of 405 amino acid residues, with an initiation codon atpositions 67-69 of the nucleotide sequence in SEQ ID NO:12, a predictedleader sequence of about 18 amino acid residues, and a deduced molecularweight of about 46 kDa. The amino acid sequence of the predicted maturePAPAI is shown in SEQ ID NO:13 from amino acid residue 1 to residue 387.The PAPAI protein shown in SEQ ID NO:12 was determined by sequencing thedeposited clone. As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, as well as thevariability of cleavage sites for leaders in different known proteins,the actual PAPAI polypeptide encoded by the deposited cDNA comprisesabout 405 amino acids, but may be anywhere in the range of 385-425 aminoacids; and the actual leader sequence of this protein is about 18 aminoacids, but may be anywhere in the range of about 10 to about 26 aminoacids.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts ofthe DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 67-69 of the nucleotide sequence shown in SEQ IDNO:1; DNA molecules comprising the coding sequence for the mature PAPAIprotein shown in FIG. 1 (last 378 amino acids) (SEQ ID NO:2); DNAmolecules comprising an ORF with an initiation codon at positions 67-69of the nucleotide sequence shown in SEQ ID NO:12; DNA moleculescomprising the coding sequence for the mature PAPAI protein shown inFIG. 4 (last 387 amino acids); and DNA molecules which comprise asequence substantially different from those described above but which,due to the degeneracy of the genetic code, still encode the PAPAIprotein. Of course, the genetic code is well known in the art. Thus, itwould be routine for one skilled in the art to generate the degeneratevariants described above.

In addition, the present inventors have identified nucleic acidmolecules having nucleotide sequences related to extensive portions ofSEQ ID NO:1 and SEQ ID NO:12 which have been determined from thefollowing related cDNA clones: HPASD5OR (SEQ ID NO:10) and HBXFM84RA(SEQ ID NO:11).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the PAPAI polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97657 on Jul. 12, 1996. In a further embodiment, nucleicacid molecules are provided encoding the mature PAPAI polypeptide orfull-length PAPAI polypeptide lacking the N-terminal methionine residue.The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in SEQ ID NO:1 or the nucleotide sequenceof the PAPAI cDNA contained in the above-described deposited clone, or anucleic acid molecule having a sequence complementary to one of theabove sequences. Such isolated molecules, particularly DNA molecules,are useful as probes for gene mapping, by in situ hybridization withchromosomes, and for detecting expression of the PAPAI gene in humantissue, for instance, by Northern blot analysis.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC Deposit 97657. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC 750 mM NaCl, 75 mM trisodium citrate 50mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C. By a polynucleotide whichhybridizes to a “portion” of a polynucleotide is intended apolynucleotide (either DNA or RNA) hybridizing to at least about 15nucleotides (nt), and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably about 30-70 ntof the reference polynucleotide. These are useful as diagnostic probesand primers as discussed above and in more detail below.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 650, 700,750, 800, 850, 900, 950, 1000, 1050, or 1100 nt in length, or even tothe entire length of the reference polynucleotide, are also useful asprobes according to the present invention, as are polynucleotidescorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1 or SEQID NO:12. By a portion of a polynucleotide of “at least 20 nt inlength,” for example, is intended 20 or more contiguous nucleotides fromthe nucleotide sequence of the reference polynucleotide (e.g., thedeposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1 or SEQID NO:12). As indicated, such portions are useful diagnostically eitheras a probe according to conventional DNA hybridization techniques or asprimers for amplification of a target sequence by the polymerase chainreaction (PCR), as described, for instance, in Molecular Cloning, ALaboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F.and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, theentire disclosure of which is hereby incorporated herein by reference.

Since a PAPAI cDNA clone has been deposited and its determinednucleotide sequence is provided in SEQ ID NO:1 and SEQ ID NO:12,generating polynucleotides which hybridize to a portion of the PAPAIcDNA molecule would be routine to the skilled artisan. For example,restriction endonuclease cleavage or shearing by sonication of the PAPAIcDNA clone could easily be used to generate DNA portions of varioussizes which are polynucleotides that hybridize to a portion of the PAPAIcDNA molecule. Alternatively, the hybridizing polynucleotides of thepresent invention could be generated synthetically according to knowntechniques. Of course, a polynucleotide which hybridizes only to a polyA sequence (such as the 3′ terminal poly(A) tract of the PAPAI cDNAshown in FIG. 1 (SEQ ID NO:1) or FIG. 4 (SEQ ID NO:12)), or to acomplementary stretch of T (or U) resides, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

The invention further provides isolated nucleic acid moleculescomprising a polynucleotide encoding an epitope-bearing portion of thePAPAI protein. In particular, isolated nucleic acid molecules of thepresent invention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 6 to about 16 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 31 to about 36in SEQ ID NO:2; a polypeptide comprising amino acid residues from about46 to about 76 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 461 to about 121 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 146 to about 161 in SEQ IDNO:2; a polypeptide comprising amino acid residues from about 206 toabout 211 in SEQ ID NO:2; a polypeptide comp rising amino acid residuesfrom about 236 to about 246 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 306 to about 316 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 361 to about 366in SEQ ID NO:2; a polypeptide comprising amino acid residues from about2 to about 12 in SEQ ID NO:13; a polypeptide comprising amino acidresidues from about 27 to about 32 in SEQ ID NO:13; a polypeptidecomprising amino acid residues from about 42 to about 72 in SEQ IDNO:13; a polypeptide comprising amino acid residues from about 107 toabout 117 in SEQ ID NO:13; a polypeptide comprising amino acid residuesfrom about 142 to about 157 in SEQ ID NO:13; a polypeptide comprisingamino acid residues from about 202 to about 207 in SEQ ID NO:13; apolypeptide comprising amino acid residues from about 232 to about 242in SEQ ID NO:13; a polypeptide comprising amino acid residues from about302 to about 312 in SEQ ID NO:13; and a polypeptide comprising aminoacid residues from about 357 to about 362 in SEQ ID NO:13. Methods forgenerating such epitope-bearing portions of PAPAI are described indetail below.

As indicated, nucleic acid molecules of the present invention whichencode a PAPAI polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the mature polypeptide, by itself,the coding sequence for the mature polypeptide and additional sequences,such as those encoding the about 14 amino acid leader or secretorysequence, such as a pre-, or pro- or prepro-protein sequence; the codingsequence of the mature polypeptide, with or without the aforementionedadditional coding sequences, together with additional, non-codingsequences, including for example, but not limited to introns andnon-coding 5′ and 3′ sequences, such as the transcribed, non-translatedsequences that play a role in transcription, mRNA processing, includingsplicing and polyadenylation signals, for example—ribosome binding andstability of mRNA; an additional coding sequence which codes foradditional amino acids, such as those which provide additionalfunctionalities. Thus, the sequence encoding the polypeptide may befused to a marker sequence, such as a sequence encoding a peptide whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (Qiagen, Inc.), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein, which has been described by Wilson etal., Cell 37: 767 (1984).

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the PAPAI protein. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced bynucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the PAPAI protein or portions thereof. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are nucleic acid molecules encoding the mature PAPAIprotein having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) orFIG. 4 (SEQ ID NO:13) or the mature PAPAI amino acid sequence encoded bythe deposited cDNA clone.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical to (a) a nucleotide sequence encoding the polypeptide havingthe amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-teiminal methionine residue; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 378 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:13;(e) a nucleotide sequence encoding the polypeptide having the amino acidsequence in SEQ ID NO:13, but lacking the N-terminal methionine residue;(f) a nucleotide sequence encoding the polypeptide having the amino acidsequence at positions from about 1 to about 387 in SEQ ID NO:13; (g) anucleotide sequence encoding the polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97657;(h) a nucleotide sequence encoded by the cDNA clone contained in ATCCDeposit No. 97657; or (i) a nucleotide sequence complementary to any ofthe nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a PAPAIpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the PAPAIpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:12 or to thenucleotides sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shownin SEQ ID NO:1 or SEQ ID NO:12 or to the nucleic acid sequence of thedeposited cDNA, irrespective of whether they encode a polypeptide havingPAPAI activity. This is because even where a particular nucleic acidmolecule does not encode a polypeptide having PAPAI activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having PAPAI activityinclude, inter alia, (1) isolating the PAPAI gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe PAPAI gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern Blot analysis for detecting PAPAI mRNA expression in specifictissues.

Preferred, however, are nucleic acid molecules having sequences at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shownin SEQ ID NO:1 or SEQ ID NO:12 or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having PAPAIprotein activity. By “a polypeptide having PAPAI activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the PAPAI protein of the invention (either thefull-length protein or, preferably, the mature protein), as measured ina particular biological assay.

Assays of plasminogen activator activity are well-known to those in theart. These assays can be used to measure plasminogen activator activityof partially purified or purified native or recombinant protein. Forexample, an ¹²⁵I fibrin lysis assay can be used (Lyon, P.B. et al., TheProstate 27:179-186 (1995); Unkeless, J.C. et al., J. Exp. Med137:85-126 (1973)).

In this assay, ¹²⁵I fibrinogen is placed into 96-well, flat bottomculture plates at a concentration of 10 μg/cm² in a volume of 10-30 μl.Dried plates are exposed to 100 μl volume of RPMI medium containing 10%fetal bovine serum. Excess thrombin in the serum results in fibrinogenconversion to fibrin with a total trypsinizable radioactivity ofapproximately 60,000 counts per minute per well. Into each test well isplaced 1-5 IU of tissue plasminogen activator (Sigma, St. Louis, Mo.).Alternatively, if cells are used in place of tissue plasminogenactivator, 1×10⁵ cells, washed twice with phosphate-buffered saline, areplaced into each test well.

Into each test well is placed 1 μg of human plasminogen and 1 to 10 μgof the partially purified or purified native or recombinant testprotein, in 5-50 μl of phosphate buffered saline.

Control wells receive an equal volume (5-50 μl) of phosphate bufferedsaline. Each sample is assayed in duplicate or triplicate, withbackground values of radioactive release determined from the duplicatesample wells without plasminogen. Additional control wells include mediaalone, media plus plasminogen, media plus plasminogen and plasminogenactivator, and wells containing up to 200 μl of 0.25% bovine trypsin todetermine the maximal releasable radioactivity.

At intervals following plating, the medium is removed and radioactivityis measured by gamma counting. Plasminogen-dependent fibrinolysis isdefined as the difference in supernatant radioactivity betweenplasminogen-containing and plasminogen-free wells. Plasminogenactivator-dependent fibrinolysis is expressed as a percentage of themaximal releasable radioactivity per well. Maximal releasableradioactivity is determined by averaging the total trypsinizableradioactivity in three fibrin-coated wells. It will be apparent to oneof ordinary skill in the art that the amounts of reactants and reactantconditions may have to be modified in order to practice the assay.

PAPAI inhibits plasminogen activators such as urokinase and tissueplasminogen activator. Thus, “a polypeptide having PAPAI proteinactivity” includes polypeptides that exhibit the plasminogen activatorinhibiting activity, in the above-described assay and in adose-dependent manner. Although the degree of dose-dependent activityneed not be identical to that of the PAPAI protein, preferably, “apolypeptide having PAPAI protein activity” will exhibit substantiallysimilar dose-dependence in a given activity as compared to the PAPAIprotein (i.e., the candidate polypeptide will exhibit greater activityor not more than about tenfold less and, preferably, not more than abouttwof old less activity relative to the reference PAPAI protein).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or99% identical to the nucleic acid sequence of the deposited cDNA or thenucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:12 will encode apolypeptide “having PAPAI protein activity.” In fact, since degeneratevariants of these nucleotide sequences all encode the same polypeptide,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingPAPAI protein activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that thereare two main approaches for studying the tolerance of an amino acidsequence to change. The first method relies on the process of evolution,in which mutations are either accepted or rejected by natural selection.The second approach uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene and selections or screensto identify sequences that maintain functionality. As the authors state,these studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The authors further indicate which amino acidchanges are likely to be permissive at a certain position of theprotein. For example, most buried amino acid residues require nonpolarside chains, whereas few features of surface side chains are generallyconserved. Other such phenotypically silent substitutions are describedin Bowie, J. U. et al., Supra, and the references cited therein.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of PAPAIpolypeptides or fragments thereof by recombinant techniques.

Recombinant constructs may be introduced into host cells using wellknown techniques such infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coil lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharnacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus DISV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

In addition to the use of expression vectors in the practice of thepresent invention, the present invention further includes novelexpression vectors comprising operator and promoter elements operativelylinked to nucleotide sequences encoding a protein of interest. Oneexample of such a vector is pHE4-5 which is described in detail below.

As summarized in FIGS. 5 and 6, components of the pHE4-5 vector (SEQ IDNO:14) include: 1) a neomycinphosphotransferase gene as a selectionmarker, 2) an E. coli origin of replication, 3) a T5 phage promotersequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence,6) the lactose operon repressor gene (lacIq). The origin of replication(oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promotersequence and operator sequences were made synthe tically. Synthe ticproduction of nucleic acid sequences is well known in the art. CLONTECH95/96 Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, PaloAlto, Calif. 94303. A nucleotide sequence encoding PAPAI (SEQ ID NO:2 or13), is operatively linked to the promoter and operator by inserting thenucleotide sequence between the NdeI and Asp718 sites of the pHE4-5vector.

As noted above, the pHE4-5 vector contains a lacIq gene. LacIq is anallele of the lacI gene which confers tight regulation of the lacoperator. Amann, E. et al., Gene 69:301-315 (1988); Stark, M., Gene51:255-267 (1987). The lacIq gene encodes a repressor protein whichbinds to lac operator sequences and blocks transcription of down-stream(i.e., 3′) sequences. However, the lacIq gene product dissociates fromthe lac operator in the presence of either lactose or certain lactoseanalogs, e.g., isopropylB-D-thiogalactopyranoside (IPTG). PAPAI thus isnot produced in appreciable quantities in uninduced host cellscontaining the pHE4-5 vector. Induction of these host cells by theaddition of an agent such as IPTG, however, results in the expression ofthe PAPAI coding sequence.

The promoter/operator sequences of the pHE4-5 vector (SEQ ID NO:15)comprise a T5 phage promoter and two lac operator sequences. Oneoperator is located 5′ to the transcriptional start site and the otheris located 3′ to the same site. These operators, when present incombination with the lacIq gene product, confer tight repression ofdown-stream sequences in the absence of a lac operon inducer, e.g.,IPTG. Expression of operatively linked sequences located down-streamfrom the lac operators may be induced by the addition of a lac operoninducer, such as IPTG. Binding of a lac inducer to the lacIq proteinsresults in their release from the lac operator sequences and theinitiation of transcription of operatively linked sequences. Lac operonregulation of gene expression is reviewed in Devlin, T., TEXTBOOK OFBIOCHEMISTRY WITH CLINICAL CCORRELATIONS, 4th Edition (1997), pages802-807.

The pHE4 series of vectors contain all of the components of the pHE4-5vector except for the PAPAI coding sequence. Features of the pHE4vectors include optimized synthetic T5 phage promoter, lac operator, andShine-Delgarno sequences. Further, these sequences are also optimallyspaced so that expression of an inserted gene may be tightly regulatedand high level of expression occurs upon induction.

Among known bacterial promoters suitable for use in the production ofproteins of the present invention include the E. coli lacI and lacZpromoters, the T3 and T7 promoters, the gpt promoter, the lambda PR andPL promoters and the trp promoter. Suitable eukaryotic promoters includethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus (RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter.

The pHE4-5 vector also contains a Shine-Delgarno sequence 5′ to the AUGinitiation codon. Shine-Delgarno sequences are short sequences generallylocated about 10 nucleotides up-stream (i.e., 5′) from the AUGinitiation codon. These sequences essentially direct prokaryoticribosomes to the AUG initiation codon.

Thus, the present invention is also directed to expression vector usefulfor the production of the proteins of the present invention. This aspectof the invention is exemplified by the pHE4-5 vector (SEQ ID NO:14).

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification.

The PAPAI protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include naturally purified products, products of chemicalsynthe tic procedures, and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

PAPAI Polypeptides and Fragments

The invention further provides an isolated PAPAI polypeptide having theamino acid sequence encoded by the deposited cDNA, or the amino acidsequence in SEQ ID NO:2 or SEQ ID NO:13, or a peptide or polypeptidecomprising a portion of the above polypeptides. The terms “peptide” and“oligopeptide” are considered synonymous (as is commonly recognized) andeach term can be used interchangeably as the context requires toindicate a chain of at least to amino acids coupled by peptidyllinkages. The word “polypeptide” is used herein for chains containingmore than ten amino acid residues. All oligopeptide and polypeptideformulas or sequences herein are written from left to right and in thedirection from amino terminus to carboxy terminus.

It will be recognized in the art that some amino acid sequences of thePAPAI polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of the PAPAI polypeptidewhich show substantial PAPAI polypeptide activity or which includeregions of PAPAI protein such as the protein portions discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions (for example, substituting one hydrophilic residuefor another, but not strongly hydrophilic for strongly hydrophobic as arule). Small changes or such “neutral” amino acid substitutions willgenerally have little effect on activity.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg and replacements amongthe aromatic residues Phe, Tyr.

As indicated in detail above, further guidance concerning which aminoacid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2 or SEQ ID NO:13, or that encoded by the deposited cDNA, may be (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fuision regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the PAPAI protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Polar GlutamineAsparagine Acidic Aspartic Acid Glutamic Acid Small Alanine SerineThreonine Methionine Glycine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given PAPAIpolypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3.

Amino acids in the PAPAI protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as plasminogen activator inhibition. Sites that arecritical for plasminogen activator inhibition can also be determined bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904(1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell or a native source. Forexample, a recombinantly produced version of the PAPAI polypeptide canbe substantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the PAPAI polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader; the maturepolypeptide encoded by the deposited the cDNA minus the leader (i.e.,the mature protein); a polypeptide comprising amino acids about −14 toabout 378 in SEQ ID NO:2; a polypeptide comprising amino acids about −13to about 378 in SEQ ID NO:2; a polypeptide comprising amino acids about1 to about 378 in SEQ ID NO:2; a polypeptide comprising amino acidsabout −18 to about 387 in SEQ ID NO:13; a polypeptide comprising aminoacids about −17 to about 387 in SEQ ID NO:13; a polypeptide comprisingamino acids about 1 to about 387 in SEQ ID NO:13; as well aspolypeptides at least 95% identical, more preferably at least 96%, 97%,98% or 99% identical to the polypeptide encoded by the deposited cDNA,to the polypeptide of SEQ ID NO:2 or SEQ ID NO:13, and also includeportions of such polypeptides with at least 30 amino acids and morepreferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a PAPAIpolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the PAPAI polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least95%, 96% 97%, 98% or 99% identical to, for instance, the amino acidsequence shown in SEQ ID NO:2 or SEQ ID NO:13 or to the amino acidsequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711. When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in diagnostic assays for detecting PAPAI protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting PAPAI protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” PAPAI protein bindingproteins which are also candidate agonist and antagonist according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. These immunogenic epitopes arebelieved to be confined to a few loci on the molecule. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen et al., Proc. Natl. Acad Sci. USA 81:3998-4002(1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthe tic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Peptides that are extremely hydrophobic and those ofsix or fewer residues generally are ineffective at inducing antibodiesthat bind to the mimicked protein; longer, soluble peptides, especiallythose containing proline residues, usually are effective. Sutcliffe etal., supra, at 661. For instance, 18 of 20 peptides designed accordingto these guidelines, containing 8-39 residues covering 75% of thesequence of the influenza virus hemagglutinin HA1 polypeptide chain,induced antibodies that reacted with the HA1 protein or intact virus;and 12/12 peptides from the MuLV polymerase and 18/18 from the rabiesglycoprotein induced antibodies that precipitated the respectiveproteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes post-translationalprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance, Wilsonet al., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies ofthe invention also are useful for purification of the mimicked protein,for instance, by adsorption chromatography using methods well known inthe art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate PAPAI-specific antibodies include: a polypeptidecomprising amino acid residues from about 6 to about 16 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 31 to about 36in SEQ ID NO:2; a polypeptide comprising amino acid residues from about46 to about 76 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 111 to about 121 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about 146 to about 161 in SEQ IDNO:2; apolypeptide comprising amino acid residues from about 206 toabout 211 in SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about 236 to about 246 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about 306 to about 316 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about 361 to about 366in SEQ ID NO:2; a polypeptide comprising amino acid residues from about2 to about 12 in SEQ ID NO:13; a polypeptide comprising amino acidresidues from about 27 to about 32 in SEQ ID NO:13; a polypeptidecomprising amino acid residues from about 42 to about 72 in SEQ IDNO:13; a polypeptide comprising amino acid residues from about 107 toabout 117 in SEQ ID NO:13; a polypeptide comprising amino acid residuesfrom about 142 to about 157 in SEQ ID NO:13; a polypeptide comprisingamino acid residues from about 202 to about 207 in SEQ ID NO:13; apolypeptide comprising amino acid residues from about 232 to about 242in SEQ ID NO:13; a polypeptide comprising amino acid residues from about302 to about 312 in SEQ ID NO:13; and a polypeptide comprising aminoacid residues from about 357 to about 362 in SEQ ID NO:13. As indicatedabove, the inventors have determined that the above polypeptidefragments are antigenic regions of the PAPAI gene.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthe sized using known methods of chemical synthe sis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) General methodfor the rapid solid-phase synthe sis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad Sci. USA 82:5131-5135. This “SimultaneousMultiple Peptide Synthe sis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods. A completely manual procedure allows 500-1000 or more synthesesto be conducted simultaneously. Houghten et al., supra, at 5134.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347-2354 (1985). Generally, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthe sized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthe sized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

As one of skill in the art will appreciate, PAPAI polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric PAPAI protein or proteinfragment alone (Fountoulakis et al., J Biochem 270:3958-3964 (1995)).

Diagnostic and Prognostic Applications of PAPAI

The present inventors believe that PAPAI is involved in inhibition ofthe plasminogen activator system. For a number of pathologic disorders,including tumor invasion and metastasis, inflammation, and complicationsof pregnancy, significantly altered levels of PAPAI gene expression canbe detected in body tissue or fluids taken from an individual havingsuch a disorder. The level of PAPAI gene expression is measured relativeto a “standard” PAPAI gene expression level, i.e., the PAPAI expressionlevel in tissue or fluids from an individual not having the pathologicdisorder. Thus, the invention provides a diagnostic method useful duringdiagnosis of a pathologic disorder, which involves assaying theexpression level of the gene encoding the PAPAI protein in tissue orbody fluid from an individual and comparing the gene expression levelwith a standard PAPAI gene expression level, whereby an increase ordecrease in the gene expression level over the standard is indicative ofa pathologic disorder.

For example, substantial alterations in PAPAI expression or activity canserve as markers of tumor invasiveness and metastasis. This is becausePAPAI regulates the fibrinolytic system. Angiogenesis, the growth of newvascular tissue, is regulated by a balance between coagulation andfibrinolysis. Angiogenesis is associated with the expansion of a primarytumor and is also required for the growth of established metastases atdistant sites (Holmgren, L. et al., Nature Medicine 1:149-153 (1995)).The level of PAI-1 in tumors indicates the level of vascularization, andhighly vascularized tumors have higher chances of invasion andmetastasis (Fazioli, F. et al., Trends Pharm. Sci. 15:25-29 (1995)).Overexpression of PAI-2 in malignant melanoma cells inhibits metastasisin vivo (Mueller, B. M. et al., Pro. Natl. Acad Sci. USA 92:205-209(1995)). The present inventors have shown that PAPAI is expressed inmyoepithelial cells surrounding normal mammary glands and in benignlesions, but not in infiltrating breast carcinomas. Thus, the inventionprovides a method for predicting whether a tumor is likely to remainstable or will invade tissue and ultimately metastasize, by measuringthe level of PAPAI expression. As a result, decisions about whethe r topursue relatively invasive clinical interventions, such as surgery,rather than relatively non-invasive interventions, such as chemotherapyand radiation the rapy, can be rationally made. The terms “tumorinvasiveness” and metastasis are well understood by those of ordinaryskill in the art. For example, see Holmgren et al., Fazioli et al., andMueller et al., supra.

Substantial alterations in PAPAI expression or activity can be used topredict whether hemorrhage is likely to occur in patients who sufferfrom hepatic illnesses. Alcoholic cirrhosis, primary biliary cirrhosis,and liver cancer are all diseases which are accompanied by hemorrhagedue to fibrinolytic bleeding. That is, the bleeding which occurs is notdue to injury, but rather is due to a dysregulated fibrinolytic system.The overall level of plasminogen activator activity represents a balancebetween the relative levels of activator and inhibitor. Changes in PAPAIactivity, or a difference in the ratio of plasminogen activator to PAPAIcan serve as indicators of imminent hemorrhage in patients who sufferfrom alcoholic cirrhosis, primary biliary cirrhosis, and liver cancer.Thus, the invention further provides a method for predicting whetherhemorrhage will occur in such patients. The terms “hemorrhage,”“alcoholic cirrhosis,” “primary biliary cirrhosis,” “liver cancer, and“fibrinolytic bleeding” are well understood by those of ordinary skillin the art. For example, see Leiper, K. et al., J. Clin. Pathol.47:214-217 (1994).

Substantial alterations in PAPAI expression or activity can be used topredict whether a patient is likely to develop preeclampsia.Preeclampsia is a clinical syndrome that affects women in the thirdtrimester of pregnancy. The syndrome is characterized by hypertensionand proteinuria. The etiology of this obstetric complication is unknown.However, it is associated with fibrin deposition in the subendotheliumof the renal glomerulus and in the decidua segments of spiral arteries.In fatal cases of eclampsia, widespread fibrin deposition has been aprominent histologic finding. Changes in PAPAI activity, or a differencein the ratio of plasminogen activator to PAPAI can serve as indicatorsof an imminent advance from the pre-eclamptic to the eclamptic state inpatients who are at risk for eclampsia. Thus, the invention furtherprovides a method for predicting whether a pre-eclamptic patient is atrisk for developing eclampsia. The term “preeclampsia” is wellunderstood by those of ordinary skill in the art. For example, see Koh,C.L. et al., Gynecol. Obstet. Invest. 35:214-221 (1993).

Alterations in PAPAI expression can be assayed at the level of messengerRNA transcription or protein expression. Suitable assay techniques aredisclosed below. Alternatively, PAPAI inhibitory activity can be assayedusing a spectrophotometric plasminogen activator inhibitor assay. Suchan assay is well-known to those of ordinary skill in the art. Forexample, see Erikkson, E. et al., Thrombosis Research 50:91-101 (1988).

By individual is intended mammalian individuals, preferably humans. By“measuring the expression level of the gene encoding the PAPAI protein”is intended qualitatively or quantitatively measuring or estimating thelevel of the PAPAI or the level of the mRNA encoding the PAPAI proteinin a first biological sample either directly (e.g., by determining orestimating absolute protein level or mRNA level) or relatively (e.g., bycomparing to the PAPAI protein level or mRNA level in a secondbiological sample). Preferably, the PAPAI protein level or mRNA level inthe first biological sample is measured or estimated and compared to astandard PAPAI protein level or mRNA level, the standard being takenfrom a second biological sample obtained from an individual not havingthe disorder or being determined by averaging levels from a populationof individuals not having the disorder. As will be appreciated in theart, once a standard PAPAI protein level or mRNA level is known, it canbe used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, a cell line, a tissue culture, or other source whichcontains PAPAI protein or mRNA, secretes mature PAPAI protein, orexpresses the PAPAI receptor. Biological samples include normal tissueor cells and tumor cells (whether malignant or benign). Biologicalsamples include body fluids, including whole blood, serum, plasma,urine, saliva, tears, pulmonary secretions, gastrointestinal secretions,fecal material, lymph fluid, synovial fluid, and cerebrospinal fluid.Methods for obtaining tissue biopsies and body fluids from mammals arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the PAPAI protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription (RT) incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada et al.,Cell 63:303-312 (1990). Briefly, total RNA is prepared from a biologicalsample as described above. For the Northern blot, the RNA is denaturedin an appropriate buffer (such as glyoxal/dimethyl sulfoxide/sodiumphosphate buffer), subjected to agarose gel electrophoresis, andtransferred onto a nitrocellulose filter. After the RNAs have beenlinked to the filter by a UV linker, the filter is prehybridized in asolution containing formamide, SSC, Denhardt's solution, denaturedsalmon sperm, SDS, and sodium phosphate buffer. PAPAI protein cDNAlabeled according to any appropriate method (such as the ³²P-multiprimedDNA labeling system (Amersham)) is used as probe. After hybridizationovernight, the filter is washed and exposed to x-ray film. cDNA for useas probe according to the present invention is described in the sectionsabove and will preferably at least 15 bp in length.

S1 mapping can be performed as described in Fujita et al., Cell49:357-367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthe sizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe PAPAI protein). Northern blot analysis can be performed as describedabove.

Preferably, levels of mRNA encoding the PAPAI protein are assayed usingthe RT-PCR method described in Makino et al., Technique 2:295-301(1990). By this method, the radioactivities of the “amplicons” in thepolyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the PAPAI protein)is quantified using an imaging analyzer. RT and PCR reaction ingredientsand conditions, reagent and gel concentrations, and labeling methods arewell known in the art. Variations on the RT-PCR method will be apparentto the skilled artisan.

Any set of oligonucleotide primers which will amplify reversetranscribed target mRNA can be used and can be designed as described inthe sections above.

Assaying PAPAI protein levels in a biological sample can occur using anyart-known method. Preferred for assaying PAPAI protein levels in abiological sample are antibody-based techniques. For example, PAPAIprotein expression in tissues can be studied with classicalimmunohistological methods. In these, the specific recognition isprovided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of PAPAI protein for Western-blot or dot/slot assay(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985)); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). In this technique, whichis based on the use of cationic solid phases, quantitation of PAPAIprotein can be accomplished using isolated PAPAI protein as a standard.This technique can also be applied to body fluids. With these samples, amolar concentration of PAPAI protein will aid to set standard values ofPAPAI protein content for different body fluids, like serum, plasma,urine, synovial fluid, spinal fluid, etc. The normal appearance of PAPAIprotein amounts can then be set using values from healthy individuals,which can be compared to those obtained from a test subject.

Other antibody-based methods useful for detecting PAPAI protein levelsinclude immunoassays, such as the enzyme linked immunoadsorbent assay(ELISA) and the radioimmunoassay (RIA). For example, PAPAIprotein-specific monoclonal antibodies can be used both as animmunoadsorbent and as an enzyme-labeled probe to detect and quantifythe PAPAI protein. The amount of PAPAI protein present in the sample canbe calculated by reference to the amount present in a standardpreparation using a linear regression computer algorithm. Such an ELISAfor detecting a tumor antigen is described in lacobelli et al., BreastCancer Research and Treatment 11:19-30 (1988). In another ELISA assay,two distinct specific monoclonal antibodies can be used to detect PAPAIprotein in a body fluid. In this assay, one of the antibodies is used asthe immunoadsorbent and the other as the enzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting PAPAI proteinwith immobilized antibody and, without washing, contacting the mixturewith the labeled antibody. The “two-step” assay involves washing beforecontacting the mixture with the labeled antibody. Other conventionalmethods may also be employed as suitable. It is usually desirable toimmobilize one component of the assay system on a support, the rebyallowing other components of the system to be brought into contact withthe component and readily removed from the sample.

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

In addition to assaying PAPAI protein levels in a biological sampleobtained from an individual, PAPAI protein can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of PAPAIprotein include those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A PAPAI protein-specific antibody or antibody portion which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined. It will be understoodin the art that the size of the subject and the imaging system used willdetermine the quantity of imaging moieties needed to produce diagnosticimages. In the case of a radioisotope moiety, for a human subject, thequantity of radioactivity injected will normally range from about 5 to20 millicuries of ^(99m)Tc. The labeled antibody or antibody portionwill then preferentially accumulate at the location of cells whichcontain PAPAI protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Portions” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, eds., S. W. Burchiel and B. A. Rhodes, MassonPublishing Inc. (1982)).

PAPAI protein-specific antibodies for use in the present invention canbe raised against the intact PAPAI protein or an antigenic polypeptideportion thereof, which may presented together with a carrier protein,such as an albumin, to an animal system (such as rabbit or mouse) or, ifit is long enough (at least about 25 amino acids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody portions (suchas, for example, Fab and F(ab′)₂ portions) which are capable ofspecifically binding to PAPAM protein. Fab and F(ab′)₂ portions lack theFc portion of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding of an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these portions arepreferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the PAPAI protein oran antigenic portion thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of PAPAI protein is prepared andpurified as described above to render it substantially free of naturalcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or PAPAI protein binding portions thereof ).Such monoclonal antibodies can be prepared using hybridoma technology(Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol.6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981)). In general, such procedures involve immunizing ananimal (preferably a mouse) with a PAPAI protein antigen or, morepreferably, with a PAPAI protein-expressing cell. Suitable cells can berecognized by their capacity to bind PAPAI protein antibody. Such cellsmay be cultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 μg/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP₂O), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225-232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the PAPAI antigen.

Alternatively, additional antibodies capable of binding to the PAPAIprotein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are the mselves antigens, and therefore it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, PAPAI protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thePAPAI protein-specific antibody can be blocked by the PAPAI proteinantigen. Such antibodies comprise anti-idiotypic antibodies to the PAPAIprotein-specific antibody and can be used to immunize an animal toinduce formation of further PAPAI protein-specific antibodies.

It will be appreciated that Fab and F(ab′)₂ and other portions of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such portions are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab portions) orpepsin (to produce F(ab′)₂ portions). Alternatively, PAPAIprotein-binding portions can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

Where in vivo imaging is used to detect enhanced levels of PAPAI proteinfor diagnosis in humans, it may be preferable to use “humanized”chimeric monoclonal antibodies. Such antibodies can be produced usinggenetic constructs derived from hybridoma cells producing the monoclonalantibodies described above. Methods for producing chimeric antibodiesare known in the art. See, for review, Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat.No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).

Further suitable labels for the PAPAI protein-specific antibodies of thepresent invention are provided below. Examples of suitable enzyme labelsinclude malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphatedehydrogenase, triose phosphate isomerase, peroxidase, alkalinephosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²EU, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled monoclonal antibody by the liver. In addition,this radio nucleotide has a more favorable gamma emission energy forimaging (Perkins et al., Eur. J. Nucl. Med. 10:296-301 (1985);Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)). For example, ¹¹¹Incoupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTAhas shown little uptake in non-tumorous tissues, particularly the liver,and the refore enhances specificity of tumor localization (Esteban etal., J. Nucl. Med. 28:861-870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an aliophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and Fe.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy et al. (Clin. Chim. Acta 70:1-31 (1976)), andSchurs et al. (Clin. Chim. Acta 81:140 (1977)). Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a PAPAI protein gene. This canbe accomplished using a variety of well known techniques and libraries,which generally are available commercially. The genomic DNA then is usedfor in situ chromosome mapping using well known techniques for thispurpose. Typically, in accordance with routine procedures for chromosomemapping, some trial and error may be necessary to identify a genomicprobe that gives a good in situ hybridization signal.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified portion.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of portions from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance In Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. This assumes 1 megabase mapping resolution and one geneper 20 kb.

Therapeutic Uses of PAPAI

As discussed above, the PAPAI-protein involved in inhibition of theplasminogen activator system and plays a role in a wide variety ofphysiologic and pathologic processes. Accordingly, the PAPAI protein hasapplication to any physiologic or pathologic disease condition in whichabnormal activity of the plasminogen activator system is implicated andhas pathological or physiological consequences. A large number ofdisease conditions are associated with modifications of the plasminogenactivator system (Kruithof et al., Thromb. Haemost. 59:7 (1988)).Examples of such disease conditions include, but are not limited to:complications of pregnancy, such as preeclampsia and intrauterine growthretardation (Halliganet al., Br. Obstet. Gyneco., 101:488 (1994);Gilabert et al., Gynecol. Obstet. Invest. 38:157(1994)); cancer (Dano etal., Fibrinolysis 8:189 (1994); Fazoli, F. et al., Trends Pharmacol.Sci. 15:25 (1994); Shinkfield et al., Fibrinolysis 6:59 (1992)); andwound healing (Schäfer et al., Amer. J. Pathol. 144:1269 (1994)).Because of the role of the plasminogen activator system in these diseasestates, inhibition of the PA system by PAPAI should provide the rapeuticbenefits to an individual suffering from one (or more) of thesephysiologic or pathologic diseases.

Given the fibrinolytic activities modulated by PAPAI, it is readilyapparent that a substantially altered level of expression of PAPAI in anindividual, compared to the standard or “normal” level, producespathological conditions such as those described above in relation todiagnosis. It will also be appreciated by one of ordinary skill that,since the PAPAI protein of the invention is translated with a leaderpeptide suitable for secretion of the mature protein from the cellswhich express PAPAI, when PAPAI protein (particularly the mature form)is added from an exogenous source to cells, tissues or the body of anindividual, the protein will exert its modulating activities on any ofits target cells of that individual. Therefore, it will be appreciatedthat conditions caused by a decrease in the standard or normal level ofPAPAI activity in an individual, can be treated by administration ofPAPAI protein. Thus, the invention also provides a method of treatmentof an individual in need of an increased level of activity comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated PAPAI polypeptide of the invention,particularly a mature form of the PAPAI protein of the invention,effective to increase the PAPAI activity level in such an individual.

For example, since plasminogen activator inhibitors inhibit tumor cellinvasion and metastasis, the invention provides a method for treating orpreventing tumor invasion and metastasis in cancers including, but notlimited to, leukemia, lung cancer, breast cancer, endometrial andovarian cancer, melanoma, and gastrointestinal cancers, includingpancreatic cancer and colorectal cancer by providing a PAPAI polypeptideto a patient in need thereof. The present inventors have shown, in ananimal model, that the presence of PAPAI alters the invasive potentialof breast cancer cells.

In addition, since plasminogen activator inhibitors inhibitfibrinolysis, the invention provides a method for treating or preventingcoagulation disorders including, but not limited to, arterial thrombi,venous thrombi, disseminated intravascular coagulation, and excessivebleeding caused by the administration of a pharmaceutical plasminogenactivator (such as urokinase or tissue plasminogen activator), byproviding a PAPAI polypeptide to a patient in need thereof.

Further, since protease inhibitors are effective antiviral agents, theinvention provides a method for treating or preventing infections causedby viruses including, but not limited to, Human Immunodeficiency Virus 1(HIV-1), HIV-2, hepatitis A, hepatitis B, hepatitis C, hepatitis E,hepatitis F, and hepatitis G, by providing a PAPAI polypeptide to apatient in need thereof.

One of ordinary skill will appreciate that effective amounts of thePAPAI polypeptides for treating an individual in need of an increasedlevel of PAPAI activity (including amounts of PAPAI polypeptideseffective for the conditions discussed above, with or without other orplasminogen activator inhibitors or other agents) can be determinedempirically for each condition where administration of PAPAI isindicated.

The polypeptide having PAPAI activity may be administered inpharmaceutical compositions in combination with one or morepharmaceutically acceptable excipients. It will be understood that, whenadministered to a human patient, the total daily usage of thepharmaceutical compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific the rapeutically effective dose level for any particularpatient will depend upon a variety of factors including the type anddegree of the response to be achieved; the specific composition an otheragent, if any, employed; the age, body weight, general health, sex anddiet of the patient; the time of administration, route ofadministration, and rate of excretion of the composition; the durationof the treatment; drugs (such as a chemotherapeutic agent) used incombination or coincidental with the specific composition; and likefactors well known in the medical arts.

The PAPAI composition to be used in the the rapy will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with PAPAI alone), the site of delivery of thePAPAI composition, the method of administration, the scheduling ofadministration, and other factors known to practitioners. The “effectiveamount” of PAPAI for purposes herein (including a PAPAI effectiveamount) is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe PAPAI administered parenterally per dose will be in the range ofabout 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, asnoted above, this will be subject to the rapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day. If given continuously,the PAPAI is typically administered at a dose rate of about 1 μg/kg/hourto about 50 μg/kg/hour, either by 1-4 injections per day or bycontinuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution or bottle solution may also be employed.

A course of PAPAI treatment to affect the fibrinolytic system appears tobe optimal if continued longer than a certain minimum number of days, 7days in the case of the mice. The length of treatment needed to observechanges and the interval following treatment for responses to occurappears to vary depending on the desired effect.

The PAPAI is also suitably administered by sustained-release systems.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277(1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release PAPAIcompositions also include liposomally entrapped PAPAI. Liposomescontaining PAPAI are prepared by methods known per se: DE 3,218,121;Epstein, et al., Proc. Natl. Acad Sci. USA 82:3688-3692 (1985); Hwang etal., Proc. Natl. Acad Sci. USA 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal PAPAI therapy.

For parenteral administration, in one embodiment, the PAPAI isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the PAPAIuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

PAPAI is typically formulated in such vehicles at a concentration ofabout 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3to 8. It will be understood that the use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofPAPAI salts.

PAPAI to be used for the rapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic PAPAIcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

PAPAI ordinarily will be stored in unit or multi-dose containers, forexample, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous PAPAI solution, and the resultingmixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized PAPAI using bacteriostaticWater-for-Injection.

Dosaging may also be arranged in a patient specific manner to provide apredetermined concentration of an PAPAI activity in the blood, asdetermined by an RIA technique, for instance. Thus patient dosaging maybe adjusted to achieve regular on-going trough blood levels, as measuredby RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500ng/ml.

Pharmaceutical compositions of the invention may be administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The PAPAI polypeptide may also be employed in accordance with thepresent invention by expression of such polypeptides in vivo or ex vivo,which is often referred to as “gene the rapy”. Gene the rapy with thePAPAI polypeptide is accomplished by introduction of a recombinantvector containing a polynucleotide encoding the PAPAI polypeptide intocells, either in vivo or ex vivo. Methods for preparing recombinantvectors are well known in the art and described, for example, inMolecular Cloning, A Laboratory Manual, 2nd. edition, edited bySambrook, J., Fritsch, E. F. and Maniatis, T., (1989), Cold SpringHarbor Laboratory Press.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by infection with arecombinant viral particle encoding a polypeptide of the presentinvention. Cells may also be engineered by other techniques, includingintroduction of recombinant plasmid vectors by means of liposomes,electroporation, microinjection, or calcium phosphate precipitation.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Arecombinant viral or plasmid vector containing the polynucleotideencoding the PAPAI polypeptide is introduced into the target cell. Viralvectors useful in the invention include retroviral vectors, adenoviralvectors, HSV-based vector systems, vaccinia vectors, papovaviruses, andadeno-associated virus.

Delivery of retroviral vectors can be accomplished either by directinfection of target; or by injection of a viral producing cell line forreplication-deficient retroviruses, which provides a continuous sourceof vector particles. Plasmid vectors can be administered in vivo byliposomes, direct injection, and receptor-mediated nucleic acidtransfer. Preferred delivery methods include those that target thespecific tissue to be treated. For example, for the treatment of breastcancer, receptor-mediated gene transfer by conjugation of the plasmidvector to estrogen via polylysine is one preferred method of delivery EP785216).

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to, theretroviral LTR; the SV40 promoter; the human cytomegalovirus (CMV)promoter; adenovirus promoters; the respiratory syncytial viruspromoter; thymidine kinase promoters; inducible promoters, such as the Mpromoter, the metallothionein promoter; heat shock promoters, andcellular promoters including, but not limited to, the histone, pol III,and β-actin promoters. Preferred promoters are those that are activeonly in the target cell. For example, for treatment of breast cancer,preferred promoters include the DF3/MUC1 promoter (Manome, Y. et al., J.Biol Che, 271: 10560-10568 (1996), the mouse mammary tumor virus longterminal repeat (Holt et al., Human Gene Therapy 7:1367-1380 (1996), andthe whey acid protein promoter (Doppler, W. et al., Mol. Endocrinol.5:1524-1632 (1991)).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Expression and Purification of PAPAI in E. coli

The DNA sequence encoding the mature PAPAI protein in the deposited cDNAclone is amplified using PCR oligonucleotide primers specific to theamino acid carboxyl terminal sequence of the PAPAI protein and to vectorsequences 3′ to the gene. Additional nucleotides containing restrictionsites to facilitate cloning are added to the 5′ and 3′ sequences,respectively.

The 5′ oligonucleotide primer has the sequence 5′ CGC CCA TGG GAA GTCAAG CCT CAA G 3′ (SEQ ID NO:5) containing the underlined Nco Irestriction site (which encodes a start ATG within the Nco I site),followed by 16 nucleotides complementary to bp 110-125 of the antisensestrand of the PAPAI protein coding sequence set out in FIG. 1 (SEQ IDNO:1).

The 3′ primer has the sequence 5′ CGC AAG CTT TCA CTT CCT TTT ATC TCCCTG 3′ (SEQ ID NO:6) containing the underlined Hind III restrictionsite, followed by 8 nucleotides complementary to bp 1250-1267 of thesense strand of the PAPAI protein coding sequence set out in FIG. 1 (SEQID NO:1), and a stop codon.

The restrictions sites are convenient to restriction enzyme sites in thebacterial expression vector pQE-60, which is used for bacterialexpression in these examples. (Qiagen, Chatsworth, Calif., 91311).

The amplified PAPAI protein DNA and the vector pQE-60 are both digestedwith Nco I and Hind II and the digested DNAs are subsequently ligatedtogether. Insertion of the PAPAI protein DNA into the pQE-60 restrictedvector places the PAPAI protein coding region downstream of and operablylinked to the vector's promoter and in-frame with an initiating AUGappropriately positioned for translation of PAPAI protein.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. colistrain M′5/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”), isused in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressing PAPAIprotein, is available commercially from Qiagen.

Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin. Plasmid DNA is isolated from resistantcolonies and the identity of the cloned DNA is confirmed by restrictionanalysis.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside (“IPTG”) are then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lac repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells are thenharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein are solubilized from the inclusionbodies into 8M urea. The 8M urea solution containing the solubilizedprotein is passed over a PD-10 column in 2X phosphate buffered saline(“PBS”), the reby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2X PBS at a concentration of 95micrograms per mL.

Analysis of the preparation by standard methods of polyacrylamide gelelectrophoresis reveals that the preparation contains about 95% monomerPAPAI protein having the expected molecular weight of approximately 44.5kDa.

Example 2 Cloning and Expression in Mammalian Cells

Most of the vectors used for the transient expression of the PAPAIprotein gene sequence in mammalian cells should carry the SV40 origin ofreplication. This allows the replication of the vector to high copynumbers in cells (e.g., COS cells) which express the T-antigen requiredfor the initiation of viral DNA synthesis. Any other suitable mammaliancell line can also be utilized for this purpose.

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g. RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).Cellular signals may, however, also be used (e.g., human actin,promoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that may be usedinclude human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1 African green monkey cells, quail QC 1-3cells, mouse L cells and Chinese hamster ovary cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrof olate reductase) is a usefulmarker to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992)). Usingthese markers, the mammalian cells are grown in selective medium and thecells with the highest resistance are selected. These cell lines containamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) cells are of ten used for the production of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology438:4470 (1985)) plus a fragment of the CMV-enhancer (Boshart et al.,Cell 41:521-530 (1985)). Multiple cloning sites, such as the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. These vectors contain, in addition to the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 2(a) Expression and Purification of Human PAPAI Protein Usingthe CHO Expression System

The vector pC1 is used for the expression of PAPAI protein. Plasmid pC1is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).Both plasmids contain the mouse DHFR gene under control of the SV40early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selectedby growing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(NTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M.,Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370,Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol. 9:64-68). Cellsgrown in increasing concentrations of MTX develop resistance to the drugby overproducing the target enzyme, DHFR, as a result of amplificationof the DHFR gene. If a second gene is linked to the DHFR gene it isusually co-amplified and over-expressed. It is state of the art todevelop cell lines carrying more than 1,000 copies of the genes.Subsequently, when the methotrexate is withdrawn, cell lines contain theamplified gene integrated into the chromosome(s).

Plasmid pC1 contains for the expression of the gene of interest a strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-4470)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Pvull,and Nrul. Behind these cloning sites the plasmid contains translationalstop codons in all three reading frames followed by the 3′ intron andthe polyadenylation site of the rat preproinsulin gene. Other highefficient promoters can also be used for the expression, e.g., the humanβ-actin promoter, the SV40 early or late promoters or the long terminalrepeats from other retroviruses, e.g., HIV and HITLVI. For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC1 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding PAPAI protein in the deposited polynucleotideis amplified using PCR oligonucleotide primers specific to the carboxylterminal sequence of the PAPAI protein and to vector sequences 3′ to thegene. Additional nucleotides containing restriction sites to facilitatecloning are added to the 5′ and 3′ sequences respectively.

The 5′ primer has the sequence 5′ CGC {umlaut over (GGA TCC)} GCC ATCATG GAC ACA ATC TTC TTG 3′ (SEQ ID NO:7) containing the underlined BamHI restriction enzyme site, followed by 16 nucleotides complementary tobp 67-84 of the antisense strand of the PAPAI protein coding sequenceset out in FIG. 1 (SEQ ID NO:1). For the full length gene, the 3′ primerhas the full length sequence CGC {umlaut over (GGT ACC)} TCA CTT CCT TTTATC TCC CTG (SEQ ID NO:8), containing the underlined Asp718 restrictionsite, followed by 8 nucleotides complementary to bp 1250-1267 of thesense strand of the PAPAI protein coding sequence set out in FIG. 1 (SEQID NO:1), and a stop codon.

The restrictions sites are convenient to restriction enzyme sites in theCHO expression vector CHO-1. The amplified PAPAI protein DNA and thevector CHO-1 both are digested with BamH I and Asp718 and the digestedDNAs subsequently ligated together. Insertion of the PAPAI protein DNAinto the BamH I/Asp718 digested vector places the PAPAI protein codingregion downstream of and operably linked to the vector's promoter. Theligation mixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037) the transformed culture is plated on ampicillin mediaplates which then are incubated to allow growth of ampicillin resistantcolonies. Plasmid DNA is isolated from resistant colonies and examinedby restriction analysis and gel sizing for the presence of thePAPAI-encoding fragment.

Transfection of CHO-DHFR-cells

Chinese hamster ovary cells lacking an active DHFR enzyme are used fortransfection. 5 μg of the expression plasmid C1 are cotransfected with0.5 μg of the plasmid pSVneo using the lipofectin method (Felgner etal., supra). The plasmid pSV2-neo contains a dominant selectable marker,the gene neo from Tn5 encoding an enzyme that confers resistance to agroup of antibiotics including G418. The cells are seeded in alpha minusMEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany)and cultivated from 10-14 days. After this period, single clones aretrypsinized and then seeded in 6-well petri dishes using differentconcentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure isrepeated until clones grow at a concentration of 100 μM.

The expression of the desired gene product is analyzed by Western blotanalysis and SDS-PAGE.

Example 2(b) Expression and Purification of Human PAPAI Protein Usingthe COS Expression System

The expression plasmid, pPAPAI HA, is made by cloning a cDNA encodingPAPAI into the expression vector pcDNAI/Amp (which can be obtained fromInvitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

A DNA fragment encoding the entire PAPAI precursor and an HA tag fusedin frame to its 3′ end is cloned into the polylinker region of thevector so that recombinant protein expression is directed by the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37: 767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is as follows. The PAPAI cDNA of thedeposited clone is amplified using primers that contain convenientrestriction sites, much as described above regarding the construction ofexpression vectors for expression of PAPAI in E coli. To facilitatedetection, purification and characterization of the expressed PAPAI, oneof the primers contains a hemagglutinin tag (“HA tag”) as describedabove.

Suitable primers include that following, which are used in this example.The 5′ primer has the sequence 5′ CGC {umlaut over (GGA TCC)} GCC ATCATG GAC ACA antisense strand of the PAPAI protein coding sequence setout in FIG. 1 (SEQ ID NO:1). For the full length gene, the 3′ primer hasthe full length sequence CGC {umlaut over (TCT AGA)} TCA AGC GTA GTC TGGGAC GTC GTA TGG GTA GGG ATT TGT CAC TCT TCC (SEQ ID NO:9), containingthe underlined Xba I restriction site, an HA tag, and 18 nucleotidescomplementary to bp 1225-1242 of the sense strand of the PAPAI proteincoding sequence set out in FIG. 1 (SEQ ID NO:1).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BamH I and Xba I and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisand gel sizing for the presence of the PAPAI-encoding fragment.

For expression of recombinant PAPAI, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).Cells are incubated under conditions for expression of PAPAI by thevector.

Expression of the PAPAI HA fusion protein is detected by radiolabellingand immunoprecipitation, using methods described in, for example Harlowet al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two daysafter transfection, the cells are labeled by incubation in mediacontaining ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE gels and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3 Cloning and Expression of the PAPAI Protein in a BaculoirusExpression System

The cDNA sequence encoding the soluble extracellular domain of PAPAIprotein receptor protein in the deposited clone is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

The 5′ primer has the sequence 5′ CGC {umlaut over (GGA TCC)} GCC ATCATG GAC ACA ATC TTC TTG 3′ (SEQ ID NO:7) containing the underlined BamHI restriction enzyme site followed by 18 bases (bp 67-84) complementaryto the antisense strand of the PAPAI protein coding sequence of FIG. 1(SEQ ID NO:1). Inserted into an expression vector, as described below,the 5′ end of the amplified fragment encoding PAPAI protein receptorprovides an efficient signal peptide. An efficient signal for initiationof translation in eukaryotic cells, as described by Kozak, M., J. Mol.Biol. 196:947-950 (1987), may be located, as appropriate, in the vectorportion of the construct.

For the full length gene, the 3′ primer has the full length sequence CGC{umlaut over (GGT ACC)} TCA CTT CCT TTT ATC TCC CTG (SEQ ID NO:8),containing the underlined Asp718 restriction followed by nucleotidescomplementary to bp 1250-1267 of the sense strand of the PAPAI proteinset out in FIG. 1 (SEQ ID NO:1), and a stop codon.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and Asp718 and againis purified on a 1% agarose gel. This fragment is designated herein F2.

The vector pA2 is used to express the PAPAI protein in the baculovirusexpression system, using standard methods, such as those described inSummers et al., A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa califomica nuclear polyhedrosisvirus (AcMNPV) followed by convenient restriction sites. For an easyselection of recombinant virus the beta-galactosidase gene from E. coliis inserted in the same orientation as the polyhedrin promoter and isfollowed by the polyadenylation signal of the polyhedrin gene. Thepolyhedrin sequences are flanked at both sides by viral sequences forcell mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2, such aspAc373, pVL941 and pAcIM1 provided, as those of skill readily willappreciate, that construction provides appropriately located signals fortranscription, translation, trafficking and the like, such as anin-frame AUG and a signal peptide, as required. Such vectors aredescribed, for example, in Luckow et al., Virology 170:31-39 (1989).Suitable vectors will be readily apparent to the skilled artisan.

The plasmid is digested with the restriction enzymes BamH I and Asp718and then is dephosphorylated using calf intestinal phosphatase, usingroutine procedures known in the art. The DNA is then isolated from a 1%agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein “V2”.

Fragment F2 and the dephosphorylated plasmid V2 are ligated togetherwith T4 DNA ligase. E. coli HB 101 cells are transformed with ligationmix and spread on culture plates. Bacteria are identified that containthe plasmid with the human PAPAI protein gene by digesting DNA fromindividual colonies using Bam HI and Asp718 and then analyzing thedigestion product by gel electrophoresis. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein as pBacPAPAI.

5 μg of plasmid pBacPAPAI is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBacPAPAI are mixed in a sterile well of a microliter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate withI ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg, Md.) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. A detailed description of a “plaque assay”of this type can also be found in the user's guide for insect cellculture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, Md., at pages 9-10.

Four days after serial dilution, the virus is added to the cells. Afterappropriate incubation, blue stained plaques are picked with the tip ofan Eppendorf pipette. The agar containing the recombinant viruses isthen resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted PAPAI protein receptor is identified by DNAanalysis including restriction mapping and sequencing. This isdesignated herein as V-PAPAI.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-PAPAI at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg, Md.). 42 hours later, 5 μCiof ³⁵S methionine and 5 MCi ³⁵S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

Example 4 Tissue Distribudon of PAPAI Protein Expression

Northern blot analysis was carried out to examine the levels ofexpression of PAPAI protein in human tissues, using methods describedby, among others, Sambrook et al, cited above. PolyA⁺ was purchased fromClontech (1020 East Meadow Circle, Palo Alto, Calif. 94303).

About 1 μg of PolyA⁺ RNA was size resolved by electrophoresis through a1% agarose gel under strongly denaturing conditions. RNA was blottedfrom the gel onto a nylon filter, and the filter then was prepared forhybridization to a detectably labeled polynucleotide probe.

As a probe to detect mRNA that encodes PAPAI protein, the antisensestrand of the coding region of the cDNA insert in the deposited clonewas labeled to a high specific activity. The cDNA was labeled by primerextension, using the Prime-It kit, available from Stratagene. Thereaction was carried out using 50 ng of the cDNA, following the standardreaction protocol as recommended by the supplier. The labeledpolynucleotide was purified away from other labeled reaction componentsby column chromatography using a Select-G-50 column, obtained from5-Prime—3-Prime, Inc. of 5603 Arapahoe Road, Boulder, Colo. 80303.

The labeled probe was hybridized to the filter, at a concentration of1,000,000 cpm/ml, as described in Kreider et al., Molecularand CellularBiology, September 1990, pp. 4846-4853. Thereafter the probe solutionwas drained and the filter was washed twice at room temperature andtwice at 65° C. with 0.1×SSC, 0.1% SDS. The filter then was then driedand exposed to film at −70° C. overnight with an intensifying screen.

The Northern blot analysis showed maximal PAPAI transcript levels inpancreas. Adipose also demonstrated a high abundant PAPAI expression. Nobands were present in other specimens analyzed, including heart, brain,kidney, liver, lung, spleen, skeletal muscle, esophagus, stomach,intestine, colon, uterus, placenta, bladder, tonsil, thymus, appendix,lymph node, gall bladder, prostate, testis and ovary.

Tissue expression of PAPAI appears to be limited. Although large amountsof transcript were detected in pancreas and adipose by Northern blot, noPAPAI transcript was detected in all other tested tissues includingliver and placenta which are the rich sources for PAI-1 and PAI-2respectively. PAPAI may function in a tissue-specific fashion as part ofan acute response to tissue remodeling.

Example 5 Expression of PAPAI Protein in Normal and Cancerous BreastCells

The expression of PAPAI in human breast cancers was investigated in avariety of human breast cancer cell lines. The RNA from human breastcancer cells was prepared using the RNA isolation kit RNAzol B(Tel-Test, Inc) based on manufacturer's instructions. Equal aliquots ofRNA were electrophoresed in a 1.2% agarose gel containing formaldehydeand transferred to a nylon membrane (Boehringer Mannheim). The membranewas pre-hybridized with ExpressHyb hybridization solution (Clontech,Inc.) at 68° C. for 30 min. The hybridization was carried out in thesame solution with ³²P-labeled PAPAI probe (1.5×10⁶ cpmlml) for 1 hourat 68° C. The membrane was then rinsed in 2×SSC containing 0.05% SDSthree times for 30 min at room temperature followed by two washes with0.1×SSC containing 0.1% SDS for 40 min at 55° C. The full-length PAPAIcDNA was isolated from the Bluescript vector followed by EcoRI and XhoIdigestion and used for preparation of a cDNA probe.

The Northern blot analysis failed to detect the PAPAI transcript in alltested breast cancer cell lines. The inability to pick up the PAPAI mRNAin breast cancer cell lines by Northern blot indicates that the PAPAIgene may be only expressed in myoepithelial cells.

In order to localize the cellular source of PAPAI expression and toassess the biological relevance of PAPAI expression in breast cancer'sprogression, in situ hybridization was performed on the fixed sectionsfrom a variety of different human breast specimens. In this example, twoaspects of PAPAI expression were examined, including the tissuelocalization (stromal versus epithelial) and the potential correlationof the loss of PAPAI expression and breast cancer malignant phenotype. A1176 bp PAPAI cDNA was inserted into pBluescript SK at unique EcoRi (5′)and XhoI (3′) sites. The digoxigenin-labeled antisense probe wasgenerated by an EcoRI cut of PAPAI cDNA plasmid and followed by T7 RNApolymerase (Boehringer Mannheim). Deparaffinized and rehydrated tissuesections were treated with 0.2 M HCl at room temperature for 20 min anddeproteinized in 0.2 mg/ml proteinaseK solution at 37° C. for 30 min.The slides were then acetylated in 0.4% acetic anhydride in 0.1Mtriethanolamine for 10 min and dehydrated in ethanol. Prehybridizationwas carry out in hybridization buffer (0.3M NaCl, 0.02 Sodium Acetate, 5mM EDTA, pH 5.0), 50% formamide, 1×Denhardt's, 10% dextran sulfate and 1mg/ml tRNA over night in a humidity chamber at 50° C. Thereafter, thesections were hybridized with 500 ng/ml DIG-labeled RNA probe in thesame components as those in the prehybridization step at 50° C. for 18h. After hybridization, the slides were then washed with 2×SSC, 0.2×SSC,0.1×SSC separately at 42° C. for 20 min and treated with Rnase A in0.1×SSC at 37° C. for 30 min. Digoxigenin detection was performed withmouse antidigoxigenin antibodies (Boehringer Mannheim) followed byincubation with biotin-conjugated secondary rabbit antimouse antibodies(DAKO). The colorimetric detections were performed with a standardindirect streptavidin-biotin immunoreaction method using the UniversalLSAB Kit (DAKO) according to the manufacture's instructions.

There was a strongly positive PAPAI hybridization in the myoepithelialcells surrounding the normal mammary glands, benign hyperplasias, andbenign fibroadenomas. The expression of PAPAI mRNA was detectable in themyoepithelial cells in all four reduction mammoplasty specimens and inthree benign lesions. In contrast, expression of PAPAI was absent infive out of five cases of infiltrating breast carcinomas. No PAPAIexpression can be detected in both normal and malignant mammaryepithelial cells and in stromal fibroblasts. In all cases a strong PAPAItranscript was found in the endothelial cells of small vessels. These insitu hybridization results are consistent with the Northern blotanalysis which showed no PAPAI expression in breast cancer cells.

The loss of PAPAI expression in the malignant breast carcinomas may bedue to the loss of putative PAPAI-producing myoepithelial cells duringthe malignant progression. It is intriguing that these data aredifferent from the previous studies that have linked excessive PAI-1 andPAI-2 expression to breast cancers as compared to normal breast (Duggan,C. et al., Br. J. Cancer 76(5):622-627 (1997); Brunner, N. et al.,Cancer Treat. Res. 71:299-309 (1994); Duffy, M. et al., Cancer 62,531-533 (1988); Duggan, C. et al., Int. J. Cancer 61:597-600 (1995);Schmitt, M. et al., Br. J. Cancer 76(3):306-311 (1997); Bianchi, E. etal, Int. J. Cancer 60:597-603 (1995); and Bouchet, C. et al., Br. J.Cancer 69:398-405 (1994)). Both in situ hybridization andimmunohistochemical staining have demonstrated a strong PAI-1 expressionin the stroma surrounding breast carcinomas or at tumor margins(Bianchi, E. et al., Int. J. Cancer 60:597-603 (1995)). In particular,it was reported that the clinical outcome of breast cancer is reverselyrelated to the levels of PAI-1 expression (Liu, G. et al., Int. J.Cancer 60:501-506 (1995)). One explanation why the elevated tumor tissuecontent of PAI-1 indicates a poor prognosis for the breast cancerpatients is that, the increased expression of PAI-1 may be reciprocallyrelated to the increased expression of u-PA and ohter proteinases duringthe tumor-mediated degradation of extracellular matrix. Therefore, thiselevated levels of PAI-1 in the stroma adjunct to the invasive breastcarcinomas may represent one of the subsequent acute host responses tothe remodeling stimuli and try to balance the local tissue degradation,but not as a causative factor. Alternatively, the high level expressionof PAI-1 in the breast cancer may favor the proposed co-expression modelthat uPA and PAI-1 have to be present in the tumor in order to achievefocalized and optimal uPA-R-mediated proteolysis and invasiveness(Dickinson, J.-L. et al., J. Biol. Chem. 270:27894-27904 (1995)).

PAI-2 most probably is acting on tumor cells in a different way thanPAI-1. In contrast to PAI-1, although the PAI-2 expression is alsoincreased in breast cancers relative to normal breast, PAI-2 expressionis detected predominantly in the malignant breast cancer cells and highlevels of PAI-2 has been proposed as a favourable prognostic marker inbreast cancer (Duggan, C. et al., Br. J. Cancer 76(5):622-627 (1997)).Since PAI-2 is mainly found as an intracellular protein, this raises apossibility as to its physicological role on protection of the cellsfrom some harmful effects of an intracellular proteinases (Schmitt, M.et al., Thromb. Haemost. 78(1):285-296 (1997)). PAI-2 is an importantfactor in cytoprotection of cell death in TNFα-induced process throughinhibition of the proteinases involved in TNFα-mediated apoptosis(Dickinson, J.-L. et al., J. Biol. Chem. 270:27894-27904 (1995)). ThisPAI-2-mediated cytoprotective role resembles the antiapoptotic action ofbcl-2 gene which is in close proximity of the PAI-2 gene on chromosome18 (Dickinson, J.-L. et al., J. Biol. Chem. 270:27894-27904 (1995);Bachmann, F., Thromb. and Haemostas. 74(1):172-179 (1995); andSternlicht, M.-D. & Barsky, S.-H, Med Hypoth. 48:37-46 (1997)). In thisregard, the increased expression of PAI-2 in breast cancer cells may bein part responsible for the less apoptotic phenotype of the malignantcancer cells. On the other hand, a secreted glycoslated form of PAI-2 isalso present (Bachmann, F., Thromb. and Haemostas. 74(1):172-179 (1995))and may contribute its anti-invasive and anti-metastatic effect throughinhibition of PA.

It is interesting to note that PAPAI expression is seen exclusively inthe myoepithelial cells which are lost during the breast cancermalignant progression. Myoepithelial cells, normally surrounding ductsof glandular organs such as breast, contribute to the synthe sis of asurrounding basement membrane and exert important paracine effects onepithelial mitogenesis and morphogenesis (Sternlicht, M.-D. & Barsky,S.-H Med. Hypoth. 48:37-46 (1997)). In normal or non-invasive benignbreast, cell-stromal contact is mediated by myoepithelial cells whichsecrete relatively low levels of matrix-degrading proteinases butrelatively high levels of maspin and various other anti-invasiveproteinase inhibitors (Sternlicht, M.-D., Safarians, S., Rivera, S.-P. &Barsky, S.-H. Lab Invest. 74 (4), 781-796 (1996)). Myoepithelial cellscan also induce differentiation of breast cancer cells (Bani, D. et al.,Br. J. Cancer 70(5):900-904 (1994)) and inhibit tumor cell invasion invitro (Sternlicht, M.-D. & Barsky, S.-H Med. Hypoth. 48:37-46 (1997)).The exclusive expression of PAPAI in myoepithelial cells of normal orbenign mammary gland may represent one of the major anti-invasiveproteinase inhibitors mediated by the host defensive myoepithelial layerin preventing breast cancer malignant progression. In this regard, theexpression of PAPAI in myoepithelial cells would create amicro-environment in the epithelial-stromal interface where theinhibitory effect of PAPAI prevents the excessive proteolytic actionsand preserve the epithelial-stromal structure integrity. It has beenrecognized that an intact myoepithelial layer, like an intactextracellular basement, can distinguish benign epithelial proliferationsand in situ carcinomas from invasive disease. The exclusive expressionof PAPAI in the myoepithelial cells may play a role as an anti-invasiveand anti-metastatic phenotype in the myoepithelial layer.

Example 6 Affect of PAPAI on Breast Cancer Progression

Reagents. Restriction enzymes, T7 polymerase, random primer DNA labelingkit, and digoxigenin-labeled nucleotides were obtained from BoehringerMannhem, Indianapolis. ³²P-dATP was purchased from Amersham.

In vitro assay for cell growth. Exponentially growing cultures ofdifferent MDA-MB-435 clones were detached with trypsin, and the trypsinwas neutralized with DMEM-10% serum. Cells were counted, diluted, andseeded in triplicate at 3,000 cells per well (24-well plate) in 1 mlDMEM-5% serum. Cell growth was measured using CellTiter 96™ AqueousNon-Radioactive cell proliferation Assay Kit (Promega).

In vitro invasion assay. The modified Boyden chamber invasion assay wasperformed as previously described (Wang, M. et al., Oncogene 14(23):2767-2774 (1997)).

Tumor growth, lymph node and lung metastasis, and microvessel counts inathymic nude mice. A nude mouse mammary fat pad tumorigenic andmetastatic assay was performed as previously described (Shi, Y.-E.,Cancer Res. 57:759-764 (1997) and Shi, Y.-E. et al., Cancer Res.57(15):3084-3091 (1997) and Wang, M. et al., Oncogene 14(23):2767-2774(1997)). Microvessel counts of primary tumors was analyzed as wepreviously described for TIMP-4 transfected MDA-MB-435 tumor model(Wang, M. et al., Oncogene 14(23):2767-2774 (1997)).

Statistical Analysis. Values were expressed as means±standard errors(SEs). Comparisons were made using the two-tailed Student's t-test.Where appropriate, the chi-squared test was used to compare proportions.

Transfection and selection of PAPAI positive clones. Since theexpression of PAPAI in the myoepithelial cells may contribute the rolesof myoepithelial layer as paracrine cellular suppression of invasion, itwas tested whether we can inhibit breast cancer invasion and metastasisby transfection of PAPAI into metastatic breast cancer cells. In orderto select a suitable breast cancer cell line for PAPAI genetransfection, a panel of breast cancer cell lines as well as normalmammary epithelial cells were screened for analysis of PAPAI expression.Northern blot analysis failed to detect the PAPAI transcript in MCF-7,T47D, MDAMB-231, MDAMB-435, MDAMB-436, ZR 75-1, Hs578t and BT549 breastcancer cell lines and NME4144 and NME4244 normal mammary epithelialcells. The inability to pick up the PAPAI mRNA in both normal andmalignant breast epithelial cells by Northern blot supports the in situhybridization data indicating the myoepithelial expression of the PAPAI.

MDA-MB-435 cell line was selected as recipient for PAPAI mediated genetransfection because: 1) it lacks detectable PAPAI transcript; and 2) itis relatively highly tumorigenic and metastatic in nude mice. HumanPAPAI cDNA was subdoned into the pCI-neo mammalian expression vector(Promega) downstream of the human cytomegalovirus promoter and enhancerto generate the pCNPAI expression vector. 40 μg pCNPAI or the controlvector pCI-neo were used for transfections. 1×10⁶ MDA-MB-435 humanbreast cancer cells were plated 24 h prior to transfection on 100-mmdishes and then incubated with IMEM supplemented with 10% FBS, 100 IU/mlpenicillin G, 100 mg/ml streptomycin and 2 mM/ml L-glutamine (GIBCO,BRL). Transfection was carried out using the calcium phosphatecoprecipitation method. DNA was removed 16 h later by replacing theincubation medium. 24h later, the cells were subcultured to five 100-mmdishes containing IMEM supplemented with 10% FBS, 100 IU/ml penicillinG, 100 mg/ml streptomycin, 2mM/ml L-glutamine and 0.8 mg/ml G418(Geneticin, GIBCO). After colonies of about 104 cells had grown, 30G418-resistant individual clones were picked and subdloned. Clones wereinitially screened by in situ hybridization on slides with a specificPAPAI antisense probe, and the positive clones were subjected toNorthern blot analysis. MDA-MB-435 subclones transfected with PAPAI cDNAwere designated PAPAI-435, and MDA-MB-435 subdlones transfected withpCI-neo were designated neo-435. Five PAI-435 clones were picked up byin situ hybridization. All five selected PAI-435 clones, PAI-435-1,PAI-435-5, PAI435-6, PAI-435-10, and PAI435-11 expressed PAI mRNAtranscripts. In contrast, none of the parental MDA-MB-435 cells or cellstransfected with plasmid vector alone produced any detectable PAPAItranscripts. No changes in morphology were observed in these clones.

Expression of plasminogen activator inhibitor activity. The anti-tPAactivity of PAPAI transfected clones was characterized. Conditionedmedia from two PAI-435 clones, one control clone and parental MDA-MB435cells were collected, concentrated, and analyzed for plasminogenactivator inhibitory activity. Although the basal level PAI-likeactivities were detected in PAPAI negative clones, the anti-tPAactivities in two PAPAI positive clones were three times higher thanthat of PAPAI negative clones. These results indicate that the PAPAItransfected clones secreted a functional PAPAI protein.

In vitro growth of PAPAI-435 cells. To determine whether PAPAIexpression affects the growth of MDA-MB-435 cells, the growth rates ofPAPAI-435-1 and PAPAI-435-10 cells were compared to that of neo-435-2and neo-435-4 cells in the monolayer culture. No significant differencesin growth rate were observed among PAPAI positive and PAPAI negativecells.

Effect of PAPAI transfection on tumorigenicity. To study the effect ofPAPAI expression on tumorigenicity, two PAPAI positive clones,PAPAI-435-1 and PAPAI-435-10, and two PAPAI negative cells, neo-435-2and neo-435-4, were tested. A pilot study was done and the data aresummarized in Table 1. After a lag phase of 7-10 days, all the 20injections in the mice given implants of PAPAI negative cells developedtumors. In contrast, only 10 injections in the mice given implants ofPAPAI positive cells developed tumors. Starting at about 27 days afterinoculation, great level of tumor necrosis was observed in tumorsderived from neo-435-1 and neo-435-10 cells. The same breast cancercells transfected with PAPAI, however, were significantly inhibited intheir tumor growth; and either no or low level of tumor necrosis wasobserved. The size of PAPAI-435-10 tumors was only 23% of that inparental neo-435-1 tumors and 21% of that in neo-435-10 tumors. Inaddition, the tumor incidence was also greatly decreased. With 10injections, only two implants developed tumors. The tumor growth ofPAPAI-435-1 cells was also significantly reduced, with 54% and 52%inhibition of tumor size observed as compared to neo435-2 and neo-435-15tumors, respectively.

Table 1 Effects of PAPAI expression on tumor sizes and tumor incidence.Tumor vol (mm³) of primary Tumor incidence Treatment Group sizeTumor/Total 435-neo-2 366.8 ± 75.26 10/10 (100%) 435-neo-4 387.38 ±91.72 10/10 (100%) PAPAI-435-1 199.54+32.88 8/10 (80%) PAPAI-435-1083.13 ± 8.41 2/10 (20%)

Discussion

In this example, the biological relevance of PAPAI in human breastcancer progression was characterized. Amino acid sequence analysisindicate that PAPAI shares considerable sequence similarity with membersof the serpin family, including PAI-1 and PAI-2. Like PAI-1, a cleavablehydrophobic signal sequence is identified for PAPAI, indicating itssecretion. PAPAI expressed in MDA-MB-435 human breast cancer cells. Asexpected, the resulting recombinant PAPAI protein possesses aninhibitory activity on PA and is secreted extracellularly, thusconfirming that the novel protein is a new member of the PAI family.

Transfection of the MDA-MB-435 cells with a PAPAI cDNA leads toincreased expression of the PAPAI transcript and anti-tPA activity whencompared to parental cell line and control cells. The reduced in vitroinvasiveness of PAPAI-435 clones compared to control cells suggests thatthe production of PAPAI altered the invasive potential of breast cancercells in this experimental model system. These results are consistentwith the previous reports on the inhibition of the invasion by PAI-1(Soff, G.-A. et al, J. Clin. Invest. 96:2593-2600 (1995)) and PAI-2(37-38). In our nude mouse model of mammary tumor, overexpression ofPAPAI resulted in several phenotypic changes: (a) there was asignificant reduction in incidence and size of primary tumors; (b)tumor-associated angiogenesis was inhibited as evidenced by the reductedmicrovessel density in the tumor; (c) the number of microscopicmetastatic lesions in the lung and lymph node was reduced. ThePAPAI-mediated in vivo tumor growth inhibition is somewhat conflictingto the in vitro similar growth rates of PAPAI-positive clones comparedto PAPAI-negative clones. The slower in vivo growth of PAPAI-435 tumorsmay be explained, in part, by PAPAI-mediated inhibition of tumorangiogenesis. Angiogenic regulatory factors have been found to modulatethe growth of human breast cancers in several orthotopic xenograftmodels. We have recently demonstrated that transfection of MDA-MB-435cells with an angiogenic factor Scatter Factor (SF) increased tumorgrowth and angiogenesis (Mignati, P., Tsuboi, R., Robbins, E. & Riflin,D.-B. J. Cell Biol. 108, 671-682 (1989)); in contrast, overexpression oftissue inhibitor of metalloproteinase 4 (TIMP4) in MDA-MB435 cellsinhibited primary tumor growth, metastasis, and tumor angiogenesis(Wang, M. et al., Oncogene 14 (23):2767-2774 (1997)). The data in thisexample indicate that despite the lack of growth inhibition of PAPAI onbreast cancer cells, PAPAI significantly inhibits tumor growth andmetastasis presumably due to its anti-angiogenic activity. In fact, uPAstimulates components of angiogenesis including chemotaxis, proteolyticmatrix degradation, and the release of basic fibroblast growth factorfrom its storage in the basement membrane (Yasunaga, C. et al., Lab.Invest. 61:689-704 (1989); Mignati, P. et al., J. Cell Biol. 108:671-682(1989); Saksela, O. & Rifkin, D.-B. J. Cell Biol. 110: 767-775 (1990);and Flaumenhaft, R. et al., J. Cell Physiol. 140:75-81 (1989)).Furthermore, both PAI-1 (Soff, G.-A. et al., J. Clin. Invest.96:2593-2600 (1995)) and PAI-2 have been demonstrated to have ananti-angiogenic activity presumably through inhibition of uPA expressedby endothelial cells in newly forming capillary sprouts.

The magnitude of the tumor-suppressing activity of PAPAI on human breastcancer is greater than that observed previously for metalloproteinaseinhibitor TIMP-4 (Wang, M. et al., Oncogene 14(23):2767-2774 (1997)) andcomparable to that observed for tumor suppressor Rb and p53 (Wang, N.-P.et al., Oncogene 8:279-288 (1993)). The exclusive expression of PAPAI inthe myoepithelial cells of normal and benign breast and the inhibitionof breast tumor growth and metastasis by PAPAI expression suggest thatPAPAI is one of the local myoepithelial-related paracrine factors thatpreserve the normal epithelial-stromal integrity and prevent themalignant progression from benign or in situ to the metastaticphenotype.

The entire disclosure of each document cited in this application ishereby incorporated herein by reference.

15 1371 base pairs nucleic acid double both cDNA not provided CDS67..1242 mat_peptide 109..1242 sig_peptide 67..108 1 GGCACGAGGGAAAACTCTAT TTTGAAAATG AATATATTTT GATTTAAACA ATACAGAGAA 60 GTCAAA ATG GACACA ATC TTC TTG TGG AGT CTT CTA TTG CTG TTT TTT 108 Met Asp Thr Ile PheLeu Trp Ser Leu Leu Leu Leu Phe Phe -14 -10 -5 GGA AGT CAA GCC TCA AGATGC TCA GCT CAA AAA AAT ACC GAA TTT GCA 156 Gly Ser Gln Ala Ser Arg CysSer Ala Gln Lys Asn Thr Glu Phe Ala 1 5 10 15 GTG GAT CTT TAT CAA GAGGTT TCC TTA TCT CAT AAG GAC AAC ATT ATA 204 Val Asp Leu Tyr Gln Glu ValSer Leu Ser His Lys Asp Asn Ile Ile 20 25 30 TTT TCA CCC CTT GGA ATA ACTTTG GTT CTT GAG ATG GTA CAA CTG GGA 252 Phe Ser Pro Leu Gly Ile Thr LeuVal Leu Glu Met Val Gln Leu Gly 35 40 45 GCC AAA GGA AAA GCA CAG CAG CAGATA AGA CAA ACT TTA AAA CAA CAG 300 Ala Lys Gly Lys Ala Gln Gln Gln IleArg Gln Thr Leu Lys Gln Gln 50 55 60 GAA ACC TCA GCT GGG GAA GAA TTT TTGGTA CTG AAG TCA TTT TGC TCT 348 Glu Thr Ser Ala Gly Glu Glu Phe Leu ValLeu Lys Ser Phe Cys Ser 65 70 75 80 GCC ATC TCA GAG AAA AAA CAA GAA TTTACA TTT AAT CTT GCC AAT GCC 396 Ala Ile Ser Glu Lys Lys Gln Glu Phe ThrPhe Asn Leu Ala Asn Ala 85 90 95 CTC TAC CTT CAA GAA GGA TTC ACT GTG AAAGAA CAG TAT CTC CAT GGC 444 Leu Tyr Leu Gln Glu Gly Phe Thr Val Lys GluGln Tyr Leu His Gly 100 105 110 AAC AAG GAA TTT TTT CAG AGT GCT ATA AAACTG GTG GAT TTT CAA GAT 492 Asn Lys Glu Phe Phe Gln Ser Ala Ile Lys LeuVal Asp Phe Gln Asp 115 120 125 GCA AAG GCT TGT GCA GAG ATG ATA AGT ACCTGG GTA GAA AGA AAA ACA 540 Ala Lys Ala Cys Ala Glu Met Ile Ser Thr TrpVal Glu Arg Lys Thr 130 135 140 GAT GGA AAA ATT AAA GAC ATG TTT TCA GGGGAA GAA TTT GGC CCT CTG 588 Asp Gly Lys Ile Lys Asp Met Phe Ser Gly GluGlu Phe Gly Pro Leu 145 150 155 160 ACT CGG CTT GTC CTG GTG AAT GCT ATTTAT TTC AAA GGA GAT TGG AAA 636 Thr Arg Leu Val Leu Val Asn Ala Ile TyrPhe Lys Gly Asp Trp Lys 165 170 175 CAG AAA TTC AGA AAA GAG GAC ACA CAGCTG ATA AAT TTT ACT AAG AAA 684 Gln Lys Phe Arg Lys Glu Asp Thr Gln LeuIle Asn Phe Thr Lys Lys 180 185 190 AAT GGT TCA ACT GTC AAA ATT CCA ATGATG AAG GCT CTT CTG AGA ACA 732 Asn Gly Ser Thr Val Lys Ile Pro Met MetLys Ala Leu Leu Arg Thr 195 200 205 AAA TAT GGT TAT TTT TCT GAA TCT TCCCTG AAC TAC CAA GTT TTA GAA 780 Lys Tyr Gly Tyr Phe Ser Glu Ser Ser LeuAsn Tyr Gln Val Leu Glu 210 215 220 TTG TCT TAC AAA GGT GAT GAA TTT AGCTTA ATT ATC ATA CTT CCT GCA 828 Leu Ser Tyr Lys Gly Asp Glu Phe Ser LeuIle Ile Ile Leu Pro Ala 225 230 235 240 GAA GGT ATG GAT ATA GAA GAA GTGGAA AAA CTA ATT ACT GCT CAA CAA 876 Glu Gly Met Asp Ile Glu Glu Val GluLys Leu Ile Thr Ala Gln Gln 245 250 255 ATC CTA AAA TGG CTC TCT GAG ATGCAA GAA GAG GAA GTA GAA ATA AGC 924 Ile Leu Lys Trp Leu Ser Glu Met GlnGlu Glu Glu Val Glu Ile Ser 260 265 270 CTC CCT AGA TTT AAA GTA GAA CAAAAA GTA GAC TTC AAA GAC GTT TTG 972 Leu Pro Arg Phe Lys Val Glu Gln LysVal Asp Phe Lys Asp Val Leu 275 280 285 TAT TCT TTG AAC ATA ACC GAG ATATTT AGT GGT GGC TGC GAC CTT TCT 1020 Tyr Ser Leu Asn Ile Thr Glu Ile PheSer Gly Gly Cys Asp Leu Ser 290 295 300 GGA ATA ACA GAT TCA TCT GAA GTGTAT GTT TCC CAA GTG ACG CAA AAA 1068 Gly Ile Thr Asp Ser Ser Glu Val TyrVal Ser Gln Val Thr Gln Lys 305 310 315 320 GTT TTC TTT GAG ATA AAT GAAGAT GGT AGT GAA GCT GCA ACA TCA ACT 1116 Val Phe Phe Glu Ile Asn Glu AspGly Ser Glu Ala Ala Thr Ser Thr 325 330 335 GGC ATA CAC ATC CCT GTG ATCATG AGT CTG GCT CAA AGC CAA TTT ATA 1164 Gly Ile His Ile Pro Val Ile MetSer Leu Ala Gln Ser Gln Phe Ile 340 345 350 GCA AAT CAT CCA TTT CTG TTTATT ATG AAG CAT AAT CCA ACA GAA TCA 1212 Ala Asn His Pro Phe Leu Phe IleMet Lys His Asn Pro Thr Glu Ser 355 360 365 ATT CTG TTT ATG GGA AGA GTGACA AAT CCC TGACACCCAG GAGATAAAAG 1262 Ile Leu Phe Met Gly Arg Val ThrAsn Pro 370 375 GAAGAGATTT AGATTCACTG TGAATGAAAA GCACAGCCTC AGAATAAAAGATGATTTCTC 1322 AAAAATAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA1371 392 amino acids amino acid linear protein not provided 2 Met AspThr Ile Phe Leu Trp Ser Leu Leu Leu Leu Phe Phe Gly Ser -14 -10 -5 1 GlnAla Ser Arg Cys Ser Ala Gln Lys Asn Thr Glu Phe Ala Val Asp 5 10 15 LeuTyr Gln Glu Val Ser Leu Ser His Lys Asp Asn Ile Ile Phe Ser 20 25 30 ProLeu Gly Ile Thr Leu Val Leu Glu Met Val Gln Leu Gly Ala Lys 35 40 45 50Gly Lys Ala Gln Gln Gln Ile Arg Gln Thr Leu Lys Gln Gln Glu Thr 55 60 65Ser Ala Gly Glu Glu Phe Leu Val Leu Lys Ser Phe Cys Ser Ala Ile 70 75 80Ser Glu Lys Lys Gln Glu Phe Thr Phe Asn Leu Ala Asn Ala Leu Tyr 85 90 95Leu Gln Glu Gly Phe Thr Val Lys Glu Gln Tyr Leu His Gly Asn Lys 100 105110 Glu Phe Phe Gln Ser Ala Ile Lys Leu Val Asp Phe Gln Asp Ala Lys 115120 125 130 Ala Cys Ala Glu Met Ile Ser Thr Trp Val Glu Arg Lys Thr AspGly 135 140 145 Lys Ile Lys Asp Met Phe Ser Gly Glu Glu Phe Gly Pro LeuThr Arg 150 155 160 Leu Val Leu Val Asn Ala Ile Tyr Phe Lys Gly Asp TrpLys Gln Lys 165 170 175 Phe Arg Lys Glu Asp Thr Gln Leu Ile Asn Phe ThrLys Lys Asn Gly 180 185 190 Ser Thr Val Lys Ile Pro Met Met Lys Ala LeuLeu Arg Thr Lys Tyr 195 200 205 210 Gly Tyr Phe Ser Glu Ser Ser Leu AsnTyr Gln Val Leu Glu Leu Ser 215 220 225 Tyr Lys Gly Asp Glu Phe Ser LeuIle Ile Ile Leu Pro Ala Glu Gly 230 235 240 Met Asp Ile Glu Glu Val GluLys Leu Ile Thr Ala Gln Gln Ile Leu 245 250 255 Lys Trp Leu Ser Glu MetGln Glu Glu Glu Val Glu Ile Ser Leu Pro 260 265 270 Arg Phe Lys Val GluGln Lys Val Asp Phe Lys Asp Val Leu Tyr Ser 275 280 285 290 Leu Asn IleThr Glu Ile Phe Ser Gly Gly Cys Asp Leu Ser Gly Ile 295 300 305 Thr AspSer Ser Glu Val Tyr Val Ser Gln Val Thr Gln Lys Val Phe 310 315 320 PheGlu Ile Asn Glu Asp Gly Ser Glu Ala Ala Thr Ser Thr Gly Ile 325 330 335His Ile Pro Val Ile Met Ser Leu Ala Gln Ser Gln Phe Ile Ala Asn 340 345350 His Pro Phe Leu Phe Ile Met Lys His Asn Pro Thr Glu Ser Ile Leu 355360 365 370 Phe Met Gly Arg Val Thr Asn Pro 375 402 amino acids aminoacid single linear protein not provided 3 Met Gln Met Ser Pro Ala LeuThr Cys Leu Val Leu Gly Leu Ala Leu 1 5 10 15 Val Phe Gly Glu Gly SerAla Val His His Pro Pro Ser Tyr Val Ala 20 25 30 His Leu Ala Ser Asp PheGly Val Arg Val Phe Gln Gln Val Ala Gln 35 40 45 Ala Ser Lys Asp Arg AsnVal Val Phe Ser Pro Tyr Gly Val Ala Ser 50 55 60 Val Leu Ala Met Leu GlnLeu Thr Thr Gly Gly Glu Thr Gln Gln Gln 65 70 75 80 Ile Gln Ala Ala MetGly Phe Lys Ile Asp Asp Lys Gly Met Ala Pro 85 90 95 Ala Leu Arg His LeuTyr Lys Glu Leu Met Gly Pro Trp Asn Lys Asp 100 105 110 Glu Ile Ser ThrThr Asp Ala Ile Phe Val Gln Arg Asp Leu Lys Leu 115 120 125 Val Gln GlyPhe Met Pro His Phe Phe Arg Leu Phe Arg Ser Thr Val 130 135 140 Lys GlnVal Asp Phe Ser Glu Val Glu Arg Ala Arg Phe Ile Ile Asn 145 150 155 160Asp Trp Val Lys Thr His Thr Lys Gly Met Ile Ser Asn Leu Leu Gly 165 170175 Lys Gly Ala Val Asp Gln Leu Thr Arg Leu Val Leu Val Asn Ala Leu 180185 190 Tyr Phe Asn Gly Gln Trp Lys Thr Pro Phe Pro Asp Ser Ser Thr His195 200 205 Arg Arg Leu Phe His Lys Ser Asp Gly Ser Thr Val Ser Val ProMet 210 215 220 Met Ala Gln Thr Asn Lys Phe Asn Tyr Thr Glu Phe Thr ThrPro Asp 225 230 235 240 Gly His Tyr Tyr Asp Ile Leu Glu Leu Pro Tyr HisGly Asp Thr Leu 245 250 255 Ser Met Phe Ile Ala Ala Pro Tyr Glu Lys GluVal Pro Leu Ser Ala 260 265 270 Leu Thr Asn Ile Leu Ser Ala Gln Leu IleSer His Trp Lys Gly Asn 275 280 285 Met Thr Arg Leu Pro Arg Leu Leu ValLeu Pro Lys Phe Ser Leu Glu 290 295 300 Thr Glu Val Asp Leu Arg Lys ProLeu Glu Asn Leu Gly Met Thr Asp 305 310 315 320 Met Phe Arg Gln Phe GlnAla Asp Phe Thr Ser Leu Ser Asp Gln Glu 325 330 335 Pro Leu His Val AlaGln Ala Leu Gln Lys Val Lys Ile Glu Val Asn 340 345 350 Glu Ser Gly ThrVal Ala Ser Ser Ser Thr Ala Val Ile Val Ser Ala 355 360 365 Arg Met AlaPro Glu Glu Ile Ile Met Asp Arg Pro Phe Leu Phe Val 370 375 380 Val ArgHis Asn Pro Thr Gly Thr Val Leu Phe Met Gly Gln Val Met 385 390 395 400Glu Pro 415 amino acids amino acid single linear protein not provided 4Met Glu Asp Leu Cys Val Ala Asn Thr Leu Phe Ala Leu Asn Leu Phe 1 5 1015 Lys His Leu Ala Lys Ala Ser Pro Thr Gln Asn Leu Phe Leu Ser Pro 20 2530 Trp Ser Ile Ser Ser Thr Met Ala Met Val Tyr Met Gly Ser Arg Gly 35 4045 Ser Thr Glu Asp Gln Met Ala Lys Val Leu Gln Phe Asn Glu Val Gly 50 5560 Ala Asn Ala Val Thr Pro Met Thr Pro Glu Asn Phe Thr Ser Cys Gly 65 7075 80 Phe Met Gln Gln Ile Gln Lys Gly Ser Tyr Pro Asp Ala Ile Leu Gln 8590 95 Ala Gln Ala Ala Asp Lys Ile His Ser Ser Phe Arg Ser Leu Ser Ser100 105 110 Ala Ile Asn Ala Ser Thr Gly Asp Tyr Leu Leu Glu Ser Val AsnLys 115 120 125 Leu Phe Gly Glu Lys Ser Ala Ser Phe Arg Glu Glu Tyr IleArg Leu 130 135 140 Cys Gln Lys Tyr Tyr Ser Ser Glu Pro Gln Ala Val AspPhe Leu Glu 145 150 155 160 Cys Ala Glu Glu Ala Arg Lys Lys Ile Asn SerTrp Val Lys Thr Gln 165 170 175 Thr Lys Gly Lys Ile Pro Asn Leu Leu ProGlu Gly Ser Val Asp Gly 180 185 190 Asp Thr Arg Met Val Leu Val Asn AlaVal Tyr Phe Lys Gly Lys Trp 195 200 205 Lys Thr Pro Phe Glu Lys Lys LeuAsn Gly Leu Tyr Pro Phe Arg Val 210 215 220 Asn Ser Ala Gln Arg Thr ProVal Gln Met Met Tyr Leu Arg Glu Lys 225 230 235 240 Leu Asn Ile Gly TyrIle Glu Asp Leu Lys Ala Gln Ile Leu Glu Leu 245 250 255 Pro Tyr Ala GlyAsp Val Ser Met Phe Leu Leu Leu Pro Asp Glu Ile 260 265 270 Ala Asp ValSer Thr Gly Leu Glu Leu Leu Glu Ser Glu Ile Thr Tyr 275 280 285 Asp LysLeu Asn Lys Trp Thr Ser Lys Asp Lys Met Ala Glu Asp Glu 290 295 300 ValGlu Val Tyr Ile Pro Gln Phe Lys Leu Glu Glu His Tyr Glu Leu 305 310 315320 Arg Ser Ile Leu Arg Ser Met Gly Met Glu Asp Ala Phe Asn Lys Gly 325330 335 Arg Ala Asn Phe Ser Gly Met Ser Glu Arg Asn Asp Leu Phe Leu Ser340 345 350 Glu Val Phe His Gln Ala Met Val Asp Val Asn Glu Glu Gly ThrGlu 355 360 365 Ala Ala Ala Gly Thr Gly Gly Val Met Thr Gly Arg Thr GlyHis Gly 370 375 380 Gly Pro Gln Phe Val Ala Asp His Pro Phe Leu Phe LeuIle Met His 385 390 395 400 Lys Ile Thr Lys Cys Ile Leu Phe Phe Gly ArgPhe Cys Ser Pro 405 410 415 25 base pairs nucleic acid single linearcDNA not provided 5 CGCCCATGGG AAGTCAAGCC TCAAG 25 30 base pairs nucleicacid single linear cDNA not provided 6 CGCAAGCTTT CACTTCCTTT TATCTCCCTG30 33 base pairs nucleic acid single linear cDNA not provided 7CGCGGATCCG CCATCATGGA CACAATCTTC TTG 33 30 base pairs nucleic acidsingle linear cDNA not provided 8 CGCGGTACCT CACTTCCTTT TATCTCCCTG 30 57base pairs nucleic acid single linear cDNA not provided 9 CGCTCTAGATCAAGCGTAGT CTGGGACGTC GTATGGGTAG GGATTTGTCA CTCTTCC 57 171 base pairsnucleic acid single linear cDNA not provided 10 NAATATATTT NNATTTAAACAATACAGAGA AGTCAAAATG GACACAATCT TCTTGTGGAG 60 TCTTCTATTG CTGTTTTTTCGAAGTCAAGC CTCANGAATG CTCAGCTGCA AAAAAATACC 120 GAATTTGCCA GTGGNATCTTTATCAAGAGG TTTCCTTCAT CTGCATAAGG N 171 515 base pairs nucleic acidsingle linear cDNA not provided 11 GGCANNANAA CAATCTNATC CAAGGACTGTGGNACTCCTG TTCCCTGCTC ATCATGTCAT 60 GGGGCATCTG CCAGGAACCA TCTTTGATGGTGTAAAAATC TTGAATACAT AAGAGGGAAA 120 TTTTAGACTT GTTAGAAAGA AGCCAAGCAATTGAGACCTT AGATAGAACT TAGAATTCTC 180 GCCGAGTTTT GTTGGGTAAT TGTTACTTCAAAAAAAAATG CAATTTCTGT TCCCTCTTTC 240 CTCCAACCAT TTATCTGGGA AGCAAGTTATTGGCAACCCA GAGCTGATTG TTGGAGCCGG 300 GGAAAATGGT GTGAAATGTG AGAAAATGTAATTGAGATAA TAAAAACAAA AGATTTTACA 360 ATATATTATC CTCTAAGTCA TCCATTAAAAAATTGGTAGC AAAAATGTGC AGTGTTTCAA 420 GACTTTTCTT TTCTTTTTTT TTNAATACCAGATTAAAGTA GACCAAAAAG TAGACTCCAA 480 AGACGTTTGG ATNCTTGAAC ATAACCGNGATATTA 515 1370 base pairs nucleic acid single linear cDNA not providedCDS 67..1281 sig_peptide 67..120 mat_peptide 121..1281 12 GGCACGAGGGAAAACTCTAT TTTGAAAATG AATATATTTT GATTTAAACA ATACAGAGAA 60 GTCAAA ATG GACACA ATC TTC TTG TGG AGT CTT CTA TTG CTG TTT TTT 108 Met Asp Thr Ile PheLeu Trp Ser Leu Leu Leu Leu Phe Phe -18 -15 -10 -5 GGA AGT CAA GCC TCAAGA TGC TCA GCT CAA AAA AAT ACC GAA TTT GCA 156 Gly Ser Gln Ala Ser ArgCys Ser Ala Gln Lys Asn Thr Glu Phe Ala 1 5 10 GTG GAT CTT TAT CAA GAGGTT TCC TTA TCT CAT AAG GAC AAC ATT ATA 204 Val Asp Leu Tyr Gln Glu ValSer Leu Ser His Lys Asp Asn Ile Ile 15 20 25 TTT TCA CCC CTT GGA ATA ACTTTG GTT CTT GAG ATG GTA CAA CTG GGA 252 Phe Ser Pro Leu Gly Ile Thr LeuVal Leu Glu Met Val Gln Leu Gly 30 35 40 GCC AAA GGA AAA GCA CAG CAG CAGATA AGA CAA ACT TTA AAA CAA CAG 300 Ala Lys Gly Lys Ala Gln Gln Gln IleArg Gln Thr Leu Lys Gln Gln 45 50 55 60 GAA ACC TCA GCT GGG GAA GAA TTTTTG GTA CTG AAG TCA TTT TGC TCT 348 Glu Thr Ser Ala Gly Glu Glu Phe LeuVal Leu Lys Ser Phe Cys Ser 65 70 75 GCC ATC TCA GAG AAA AAA CAA GAA TTTACA TTT AAT CTT GCC AAT GCC 396 Ala Ile Ser Glu Lys Lys Gln Glu Phe ThrPhe Asn Leu Ala Asn Ala 80 85 90 CTC TAC CTT CAA GAA GGA TTC ACT GTG AAAGAA CAG TAT CTC CAT GGC 444 Leu Tyr Leu Gln Glu Gly Phe Thr Val Lys GluGln Tyr Leu His Gly 95 100 105 AAC AAG GAA TTT TTT CAG AGT GCT ATA AAACTG GTG GAT TTT CAA GAT 492 Asn Lys Glu Phe Phe Gln Ser Ala Ile Lys LeuVal Asp Phe Gln Asp 110 115 120 GCA AAG GCT TGT GCA GAG ATG ATA AGT ACCTGG GTA GAA AGA AAA ACA 540 Ala Lys Ala Cys Ala Glu Met Ile Ser Thr TrpVal Glu Arg Lys Thr 125 130 135 140 GAT GGA AAA ATT AAA GAC ATG TTT TCAGGG GAA GAA TTT GGC CCT CTG 588 Asp Gly Lys Ile Lys Asp Met Phe Ser GlyGlu Glu Phe Gly Pro Leu 145 150 155 ACT CGG CTT GTC CTG GTG AAT GCT ATTTAT TTC AAA GGA GAT TGG AAA 636 Thr Arg Leu Val Leu Val Asn Ala Ile TyrPhe Lys Gly Asp Trp Lys 160 165 170 CAG AAA TTC AGA AAA GAG GAC ACA CAGCTG ATA AAT TTT ACT AAG AAA 684 Gln Lys Phe Arg Lys Glu Asp Thr Gln LeuIle Asn Phe Thr Lys Lys 175 180 185 AAT GGT TCA ACT GTC AAA ATT CCA ATGATG AAG GCT CTT CTG AGA ACA 732 Asn Gly Ser Thr Val Lys Ile Pro Met MetLys Ala Leu Leu Arg Thr 190 195 200 AAA TAT GGT TAT TTT TCT GAA TCT TCCCTG AAC TAC CAA GTT TTA GAA 780 Lys Tyr Gly Tyr Phe Ser Glu Ser Ser LeuAsn Tyr Gln Val Leu Glu 205 210 215 220 TTG TCT TAC AAA GGT GAT GAA TTTAGC TTA ATT ATC ATA CTT CCT GCA 828 Leu Ser Tyr Lys Gly Asp Glu Phe SerLeu Ile Ile Ile Leu Pro Ala 225 230 235 GAA GGT ATG GAT ATA GAA GAA GTGGAA AAA CTA ATT ACT GCT CAA CAA 876 Glu Gly Met Asp Ile Glu Glu Val GluLys Leu Ile Thr Ala Gln Gln 240 245 250 ATC CTA AAA TGG CTC TCT GAG ATGCAA GAA GAG GAA GTA GAA ATA AGC 924 Ile Leu Lys Trp Leu Ser Glu Met GlnGlu Glu Glu Val Glu Ile Ser 255 260 265 CTC CCT AGA TTT AAA GTA GAA CAAAAA GTA GAC TTC AAA GAC GTT TTG 972 Leu Pro Arg Phe Lys Val Glu Gln LysVal Asp Phe Lys Asp Val Leu 270 275 280 TAT TCT TTG AAC ATA ACC GAG ATATTT AGT GGT GGC TGC GAC CTT TCT 1020 Tyr Ser Leu Asn Ile Thr Glu Ile PheSer Gly Gly Cys Asp Leu Ser 285 290 295 300 GGA ATA ACA GAT TCA TCT GAAGTG TAT GTT TCC CAA GTG ACG CAA AAA 1068 Gly Ile Thr Asp Ser Ser Glu ValTyr Val Ser Gln Val Thr Gln Lys 305 310 315 GTT TTC TTT GAG ATA AAT GAAGAT GGT AGT GAA GCT GCA ACA TCA ACT 1116 Val Phe Phe Glu Ile Asn Glu AspGly Ser Glu Ala Ala Thr Ser Thr 320 325 330 GGC ATA CAC ATC CCT GTG ATCATG AGT CTG GCT CAA AGC CAA TTT ATA 1164 Gly Ile His Ile Pro Val Ile MetSer Leu Ala Gln Ser Gln Phe Ile 335 340 345 GCA AAT CAT CCA TTT CTG TTTATT ATG AAG CAT AAT CCA ACA GAA TCA 1212 Ala Asn His Pro Phe Leu Phe IleMet Lys His Asn Pro Thr Glu Ser 350 355 360 ATT CTG TTT ATG GGA AGA GTGACA AAT CCT GAC ACC CAG GAG ATA AAA 1260 Ile Leu Phe Met Gly Arg Val ThrAsn Pro Asp Thr Gln Glu Ile Lys 365 370 375 380 GGA AGA GAT TTA GAT TCACTG TGAATGAAAA GCACAGCCTC AGAATAAAAG 1311 Gly Arg Asp Leu Asp Ser Leu385 ATGATTTCTC AAAAATAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA1370 405 amino acids amino acid linear protein not provided 13 Met AspThr Ile Phe Leu Trp Ser Leu Leu Leu Leu Phe Phe Gly Ser -18 -15 -10 -5Gln Ala Ser Arg Cys Ser Ala Gln Lys Asn Thr Glu Phe Ala Val Asp 1 5 10Leu Tyr Gln Glu Val Ser Leu Ser His Lys Asp Asn Ile Ile Phe Ser 15 20 2530 Pro Leu Gly Ile Thr Leu Val Leu Glu Met Val Gln Leu Gly Ala Lys 35 4045 Gly Lys Ala Gln Gln Gln Ile Arg Gln Thr Leu Lys Gln Gln Glu Thr 50 5560 Ser Ala Gly Glu Glu Phe Leu Val Leu Lys Ser Phe Cys Ser Ala Ile 65 7075 Ser Glu Lys Lys Gln Glu Phe Thr Phe Asn Leu Ala Asn Ala Leu Tyr 80 8590 Leu Gln Glu Gly Phe Thr Val Lys Glu Gln Tyr Leu His Gly Asn Lys 95100 105 110 Glu Phe Phe Gln Ser Ala Ile Lys Leu Val Asp Phe Gln Asp AlaLys 115 120 125 Ala Cys Ala Glu Met Ile Ser Thr Trp Val Glu Arg Lys ThrAsp Gly 130 135 140 Lys Ile Lys Asp Met Phe Ser Gly Glu Glu Phe Gly ProLeu Thr Arg 145 150 155 Leu Val Leu Val Asn Ala Ile Tyr Phe Lys Gly AspTrp Lys Gln Lys 160 165 170 Phe Arg Lys Glu Asp Thr Gln Leu Ile Asn PheThr Lys Lys Asn Gly 175 180 185 190 Ser Thr Val Lys Ile Pro Met Met LysAla Leu Leu Arg Thr Lys Tyr 195 200 205 Gly Tyr Phe Ser Glu Ser Ser LeuAsn Tyr Gln Val Leu Glu Leu Ser 210 215 220 Tyr Lys Gly Asp Glu Phe SerLeu Ile Ile Ile Leu Pro Ala Glu Gly 225 230 235 Met Asp Ile Glu Glu ValGlu Lys Leu Ile Thr Ala Gln Gln Ile Leu 240 245 250 Lys Trp Leu Ser GluMet Gln Glu Glu Glu Val Glu Ile Ser Leu Pro 255 260 265 270 Arg Phe LysVal Glu Gln Lys Val Asp Phe Lys Asp Val Leu Tyr Ser 275 280 285 Leu AsnIle Thr Glu Ile Phe Ser Gly Gly Cys Asp Leu Ser Gly Ile 290 295 300 ThrAsp Ser Ser Glu Val Tyr Val Ser Gln Val Thr Gln Lys Val Phe 305 310 315Phe Glu Ile Asn Glu Asp Gly Ser Glu Ala Ala Thr Ser Thr Gly Ile 320 325330 His Ile Pro Val Ile Met Ser Leu Ala Gln Ser Gln Phe Ile Ala Asn 335340 345 350 His Pro Phe Leu Phe Ile Met Lys His Asn Pro Thr Glu Ser IleLeu 355 360 365 Phe Met Gly Arg Val Thr Asn Pro Asp Thr Gln Glu Ile LysGly Arg 370 375 380 Asp Leu Asp Ser Leu 385 3974 base pairs nucleic acidboth both cDNA not provided 14 GGTACCTAAG TGAGTAGGGC GTCCGATCGACGGACGCCTT TTTTTTGAAT TCGTAATCAT 60 GGTCATAGCT GTTTCCTGTG TGAAATTGTTATCCGCTCAC AATTCCACAC AACATACGAG 120 CCGGAAGCAT AAAGTGTAAA GCCTGGGGTGCCTAATGAGT GAGCTAACTC ACATTAATTG 180 CGTTGCGCTC ACTGCCCGCT TTCCAGTCGGGAAACCTGTC GTGCCAGCTG CATTAATGAA 240 TCGGCCAACG CGCGGGGAGA GGCGGTTTGCGTATTGGGCG CTCTTCCGCT TCCTCGCTCA 300 CTGACTCGCT GCGCTCGGTC GTTCGGCTGCGGCGAGCGGT ATCAGCTCAC TCAAAGGCGG 360 TAATACGGTT ATCCACAGAA TCAGGGGATAACGCAGGAAA GAACATGTGA GCAAAAGGCC 420 AGCAAAAGGC CAGGAACCGT AAAAAGGCCGCGTTGCTGGC GTTTTTCCAT AGGCTCCGCC 480 CCCCTGACGA GCATCACAAA AATCGACGCTCAAGTCAGAG GTGGCGAAAC CCGACAGGAC 540 TATAAAGATA CCAGGCGTTT CCCCCTGGAAGCTCCCTCGT GCGCTCTCCT GTTCCGACCC 600 TGCCGCTTAC CGGATACCTG TCCGCCTTTCTCCCTTCGGG AAGCGTGGCG CTTTCTCATA 660 GCTCACGCTG TAGGTATCTC AGTTCGGTGTAGGTCGTTCG CTCCAAGCTG GGCTGTGTGC 720 ACGAACCCCC CGTTCAGCCC GACCGCTGCGCCTTATCCGG TAACTATCGT CTTGAGTCCA 780 ACCCGGTAAG ACACGACTTA TCGCCACTGGCAGCAGCCAC TGGTAACAGG ATTAGCAGAG 840 CGAGGTATGT AGGCGGTGCT ACAGAGTTCTTGAAGTGGTG GCCTAACTAC GGCTACACTA 900 GAAGAACAGT ATTTGGTATC TGCGCTCTGCTGAAGCCAGT TACCTTCGGA AAAAGAGTTG 960 GTAGCTCTTG ATCCGGCAAA CAAACCACCGCTGGTAGCGG TGGTTTTTTT GTTTGCAAGC 1020 AGCAGATTAC GCGCAGAAAA AAAGGATCTCAAGAAGATCC TTTGATCTTT TCTACGGGGT 1080 CTGACGCTCA GTGGAACGAA AACTCACGTTAAGGGATTTT GGTCATGAGA TTATCGTCGA 1140 CAATTCGCGC GCGAAGGCGA AGCGGCATGCATTTACGTTG ACACCATCGA ATGGTGCAAA 1200 ACCTTTCGCG GTATGGCATG ATAGCGCCCGGAAGAGAGTC AATTCAGGGT GGTGAATGTG 1260 AAACCAGTAA CGTTATACGA TGTCGCAGAGTATGCCGGTG TCTCTTATCA GACCGTTTCC 1320 CGCGTGGTGA ACCAGGCCAG CCACGTTTCTGCGAAAACGC GGGAAAAAGT GGAAGCGGCG 1380 ATGGCGGAGC TGAATTACAT TCCCAACCGCGTGGCACAAC AACTGGCGGG CAAACAGTCG 1440 TTGCTGATTG GCGTTGCCAC CTCCAGTCTGGCCCTGCACG CGCCGTCGCA AATTGTCGCG 1500 GCGATTAAAT CTCGCGCCGA TCAACTGGGTGCCAGCGTGG TGGTGTCGAT GGTAGAACGA 1560 AGCGGCGTCG AAGCCTGTAA AGCGGCGGTGCACAATCTTC TCGCGCAACG CGTCAGTGGG 1620 CTGATCATTA ACTATCCGCT GGATGACCAGGATGCCATTG CTGTGGAAGC TGCCTGCACT 1680 AATGTTCCGG CGTTATTTCT TGATGTCTCTGACCAGACAC CCATCAACAG TATTATTTTC 1740 TCCCATGAAG ACGGTACGCG ACTGGGCGTGGAGCATCTGG TCGCATTGGG TCACCAGCAA 1800 ATCGCGCTGT TAGCGGGCCC ATTAAGTTCTGTCTCGGCGC GTCTGCGTCT GGCTGGCTGG 1860 CATAAATATC TCACTCGCAA TCAAATTCAGCCGATAGCGG AACGGGAAGG CGACTGGAGT 1920 GCCATGTCCG GTTTTCAACA AACCATGCAAATGCTGAATG AGGGCATCGT TCCCACTGCG 1980 ATGCTGGTTG CCAACGATCA GATGGCGCTGGGCGCAATGC GCGCCATTAC CGAGTCCGGG 2040 CTGCGCGTTG GTGCGGATAT CTCGGTAGTGGGATACGACG ATACCGAAGA CAGCTCATGT 2100 TATATCCCGC CGTTAACCAC CATCAAACAGGATTTTCGCC TGCTGGGGCA AACCAGCGTG 2160 GACCGCTTGC TGCAACTCTC TCAGGGCCAGGCGGTGAAGG GCAATCAGCT GTTGCCCGTC 2220 TCACTGGTGA AAAGAAAAAC CACCCTGGCGCCCAATACGC AAACCGCCTC TCCCCGCGCG 2280 TTGGCCGATT CATTAATGCA GCTGGCACGACAGGTTTCCC GACTGGAAAG CGGGCAGTGA 2340 GCGCAACGCA ATTAATGTAA GTTAGCGCGAATTGTCGACC AAAGCGGCCA TCGTGCCTCC 2400 CCACTCCTGC AGTTCGGGGG CATGGATGCGCGGATAGCCG CTGCTGGTTT CCTGGATGCC 2460 GACGGATTTG CACTGCCGGT AGAACTCCGCGAGGTCGTCC AGCCTCAGGC AGCAGCTGAA 2520 CCAACTCGCG AGGGGATCGA GCCCGGGGTGGGCGAAGAAC TCCAGCATGA GATCCCCGCG 2580 CTGGAGGATC ATCCAGCCGG CGTCCCGGAAAACGATTCCG AAGCCCAACC TTTCATAGAA 2640 GGCGGCGGTG GAATCGAAAT CTCGTGATGGCAGGTTGGGC GTCGCTTGGT CGGTCATTTC 2700 GAACCCCAGA GTCCCGCTCA GAAGAACTCGTCAAGAAGGC GATAGAAGGC GATGCGCTGC 2760 GAATCGGGAG CGGCGATACC GTAAAGCACGAGGAAGCGGT CAGCCCATTC GCCGCCAAGC 2820 TCTTCAGCAA TATCACGGGT AGCCAACGCTATGTCCTGAT AGCGGTCCGC CACACCCAGC 2880 CGGCCACAGT CGATGAATCC AGAAAAGCGGCCATTTTCCA CCATGATATT CGGCAAGCAG 2940 GCATCGCCAT GGGTCACGAC GAGATCCTCGCCGTCGGGCA TGCGCGCCTT GAGCCTGGCG 3000 AACAGTTCGG CTGGCGCGAG CCCCTGATGCTCTTCGTCCA GATCATCCTG ATCGACAAGA 3060 CCGGCTTCCA TCCGAGTACG TGCTCGCTCGATGCGATGTT TCGCTTGGTG GTCGAATGGG 3120 CAGGTAGCCG GATCAAGCGT ATGCAGCCGCCGCATTGCAT CAGCCATGAT GGATACTTTC 3180 TCGGCAGGAG CAAGGTGAGA TGACAGGAGATCCTGCCCCG GCACTTCGCC CAATAGCAGC 3240 CAGTCCCTTC CCGCTTCAGT GACAACGTCGAGCACAGCTG CGCAAGGAAC GCCCGTCGTG 3300 GCCAGCCACG ATAGCCGCGC TGCCTCGTCCTGCAGTTCAT TCAGGGCACC GGACAGGTCG 3360 GTCTTGACAA AAAGAACCGG GCGCCCCTGCGCTGACAGCC GGAACACGGC GGCATCAGAG 3420 CAGCCGATTG TCTGTTGTGC CCAGTCATAGCCGAATAGCC TCTCCACCCA AGCGGCCGGA 3480 GAACCTGCGT GCAATCCATC TTGTTCAATCATGCGAAACG ATCCTCATCC TGTCTCTTGA 3540 TCAGATCTTG ATCCCCTGCG CCATCAGATCCTTGGCGGCA AGAAAGCCAT CCAGTTTACT 3600 TTGCAGGGCT TCCCAACCTT ACCAGAGGGCGCCCCAGCTG GCAATTCCGG TTCGCTTGCT 3660 GTCCATAAAA CCGCCCAGTC TAGCTATCGCCATGTAAGCC CACTGCAAGC TACCTGCTTT 3720 CTCTTTGCGC TTGCGTTTTC CCTTGTCCAGATAGCCCAGT AGCTGACATT CATCCGGGGT 3780 CAGCACCGTT TCTGCGGACT GGCTTTCTACGTGTTCCGCT TCCTTTAGCA GCCCTTGCGC 3840 CCTGAGTGCT TGCGGCAGCG TGAAGCTTAAAAAACTGCAA AAAATAGTTT GACTTGTGAG 3900 CGGATAACAA TTAAGATGTA CCCAATTGTGAGCGGATAAC AATTTCACAC ATTAAAGAGG 3960 AGAAATTACA TATG 3974 112 basepairs nucleic acid both both cDNA not provided 15 AAGCTTAAAA AACTGCAAAAAATAGTTTGA CTTGTGAGCG GATAACAATT AAGATGTACC 60 CAATTGTGAG CGGATAACAATTTCACACAT TAAAGAGGAG AAATTACATA TG 112

What is claimed is:
 1. An isolated polynucleotide, comprising a nucleicacid encoding an amino acid sequence at least 95% identical to areference amino acid sequence selected from the group consisting of: (a)amino acids −14 to 378 of SEQ ID NO:2; (b) amino acids −13 to 378 of SEQID NO:2; and (c) amino acids 1 to 378 of SEQ ID NO:2; wherein saidpolynucleotide encodes a polypeptide which binds to an antibody thatspecifically binds a polypeptide consisting of amino acids 1 to 378 ofSEQ ID NO:2.
 2. The isolated polynucleotide of claim 1, wherein saidamino acid sequence is at least 97% identical to said reference aminoacid sequence.
 3. The isolated polynucleotide of claim 1, wherein saidreference amino acid sequence is (a).
 4. The isolated polynucleotide ofclaim 3, wherein said nucleic acid encodes amino acids −14 to 378 of SEQID NO:2.
 5. The isolated polynucleotide of claim 1, wherein saidreference amino acid sequence is (b).
 6. The isolated polynucleotide ofclaim 5, wherein said nucleic acid encodes amino acids −13 to 378 of SEQID NO:2.
 7. The isolated polynucleotide of claim 1, wherein saidreference amino acid sequence is (c).
 8. The isolated polynucleotide ofclaim 7, wherein said nucleic acid encodes amino acids 1 to 378 of SEQID NO:2.
 9. The isolated polynucleotide of claim 1, which is DNA. 10.The isolated polynucleotide of claim 1, which is RNA.
 11. Thepolynucleotide of claim 1, further comprising a heterologouspolynucleotide.
 12. The polynucleotide of claim 11, wherein saidheterologous polynucleotide encodes a heterologous polypeptide.
 13. Amethod of producing a vector that comprises inserting the polynucleotideof claim 1 into a vector.
 14. A vector comprising the polynucleotide ofclaim
 1. 15. The vector of claim 14, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence.
 16. A hostcell comprising the polynucleotide of claim
 1. 17. The host cell ofclaim 16, wherein said polynucleotide is operably associated with aheterologous regulatory sequence.
 18. A method of producing apolypeptide comprising culturing the host cell of claim 17 underconditions such that said polypeptide is expressed, and recovering saidpolypeptide.
 19. A composition comprising the polynucleotide of claim 1and a pharmaceutically acceptable carrier.
 20. An isolatedpolynucleotide, comprising a nucleic acid encoding an amino acidsequence at least 95% identical to a reference amino acid sequenceselected from the group consisting of: (a) the full length polypeptideas encoded by the cDNA clone contained in ATCC Deposit No. 97656; and(b) the mature polypeptide as encoded by the cDNA clone contained inATCC Deposit No. 97656; wherein said polynucleotide encodes apolypeptide which binds to an antibody that specifically binds apolypeptide consisting of the mature amino acid sequence as encoded bythe cDNA clone contained in ATCC Deposit No.
 97656. 21. The isolatedpolynucleotide of claim 20, wherein said amino acid sequence is at least97% identical to said reference amino acid sequence.
 22. The isolatedpolynucleotide of claim 20, wherein said reference amino acid is (a).23. The isolated polynucleotide of claim 22, wherein said nucleic acidencodes the full length polypeptide as encoded by the cDNA clonecontained in ATCC Deposit No.
 97656. 24. The isolated polynucleotide ofclaim 20, wherein said reference amino acid is (b).
 25. The isolatedpolynucleotide of claim 24, wherein said nucleic acid encodes the maturepolypeptide as encoded by the cDNA clone contained in ATCC Deposit No.97656.
 26. The isolated polynucleotide of claim 20, which is DNA. 27.The isolated polynucleotide of claim 20, which is RNA.
 28. Thepolynucleotide of claim 20, further comprising a heterologouspolynucleotide.
 29. The polynucleotide of claim 28, wherein saidheterologous polynucleotide encodes a heterologous polypeptide.
 30. Amethod of producing a vector that comprises inserting the polynucleotideof claim 20 into a vector.
 31. A vector comprising the polynucleotide ofclaim
 20. 32. The vector of claim 31, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence.
 33. A hostcell comprising the polynucleotide of claim
 20. 34. The host cell ofclaim 33, wherein said polynucleotide is operably associated with aheterologous regulatory sequence.
 35. A method of producing apolypeptide comprising culturing the host cell of claim 34 underconditions such that said polypeptide is expressed, and recovering saidpolypeptide.
 36. A composition comprising the polynucleotide of claim 20and a pharmaceutically acceptable carrier.
 37. An isolatedpolynucleotide, comprising a nucleic acid encoding an amino acidsequence at least 95% identical to a reference amino acid sequenceselected from the group consisting of: (a) amino acids −18 to 387 of SEQID NO:13; (b) amino acids −17 to 387 of SEQ ID NO:13; and (c) aminoacids 1 to 387 of SEQ ID NO:13; wherein said polynucleotide encodes apolypeptide which binds to an antibody that specifically binds apolypeptide consisting of amino acids 1 to 378 of SEQ ID NO:13.
 38. Theisolated polynucleotide of claim 37, wherein said amino acid sequence isat least 97% identical to said reference amino acid sequence.
 39. Theisolated polynucleotide of claim 37, wherein said reference amino acidsequence is (a).
 40. The isolated polynucleotide of claim 39, whereinsaid nucleic acid encodes amino acids −18 to 387 of SEQ ID NO:13. 41.The isolated polynucleotide of claim 37, wherein said reference aminoacid sequence is (b).
 42. The isolated polynucleotide of claim 41,wherein said nucleic acid encodes amino acids −17 to 387 of SEQ IDNO:13.
 43. The isolated polynucleotide of claim 37, wherein saidreference amino acid sequence is (c).
 44. The isolated polynucleotide ofclaim 43, wherein said nucleic acid encodes amino acids 1 to 387 of SEQID NO:13.
 45. The isolated polynucleotide of claim 37, which is DNA. 46.The isolated polynucleotide of claim 37, which is RNA.
 47. Thepolynucleotide of claim 7, further comprising a heterologouspolynucleotide.
 48. The polynucleotide of claim 47, wherein saidheterologous polynucleotide encodes a heterologous polypeptide.
 49. Amethod of producing a vector that comprises inserting the polynucleotideof claim 37 into a vector.
 50. A vector comprising the polynucleotide ofclaim
 37. 51. The vector of claim 50, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence.
 52. A hostcell comprising the polynucleotide of claim
 37. 53. The host cell ofclaim 52, wherein said polynucleotide is operably associated with aheterologous regulatory sequence.
 54. A method of producing apolypeptide that comprises culturing the host cell of claim 53 underconditions such that said polypeptide is expressed, and recovering saidpolypeptide.
 55. A composition comprising the polynucleotide of claim 37and a pharmaceutically acceptable carrier.
 56. An isolatedpolynucleotide comprising a nucleic acid encoding amino acids 9 to 76 ofSEQ ID NO:2.
 57. The isolated polynucleotide of claim 56, which is DNA.58. The isolated polynucleotide of claim 56, which is RNA.
 59. Thepolynucleotide of claim 56, further comprising a heterologouspolynucleotide.
 60. The polynucleotide of claim 59, wherein saidheterologous polynucleotide encodes a heterologous polypeptide.
 61. Amethod of producing a vector that comprises inserting the polynucleotideof claim 56 into a vector.
 62. A vector comprising the polynucleotide ofclaim
 56. 63. The vector of claim 62, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence.
 64. A hostcell comprising the polynucleotide of claim
 56. 65. The host cell ofclaim 64, wherein said polynucleotide is operably associated with aheterologous regulatory sequence.
 66. A method of producing apolypeptide that comprises culturing the host cell of claim 65 underconditions such that said polypeptide is expressed, and recovering saidpolypeptide.
 67. A composition comprising the polynucleotide of claim 56and a pharmaceutically acceptable carrier.
 68. An isolatedpolynucleotide comprising a first nucleic acid which hybridizes (i) at42° C. in a solution consisting of 50% formamide, 5×SSC, 50 mM sodiumphosphate, 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA; (ii) followed by washing in asolution consisting of 0.1×SSC at 65° C.; to a second nucleic acidhaving the nucleotide sequence of the coding region of SEQ ID NO:1 orSEQ ID NO:12 or the complement thereof; wherein said first nucleic acidencodes a polypeptide which inhibits a plasminogen activator.
 69. Thepolynucleotide of claim 68, which is DNA.
 70. The polynucleotide ofclaim 68, which is RNA.
 71. The polynucleotide of claim 68, furthercomprising a heterologous polynucleotide.
 72. The polynucleotide ofclaim 71, wherein said heterologous polynucleotide encodes aheterologous polypeptide.
 73. A method of producing a vector thatcomprises inserting the polynucleotide of claim 68 into a vector.
 74. Avector comprising the polynucleotide of claim
 73. 75. The vector ofclaim 74, wherein said polynucleotide is operably associated with aheterologous regulatory sequence.
 76. A host cell comprising thepolynucleotide of claim
 68. 77. The host cell of claim 76, wherein saidpolynucleotide is operably associated with a heterologous regulatorysequence.
 78. A method of producing a polypeptide that comprisesculturing the host cell of claim 77 under conditions such that saidpolypeptide is expressed, and recovering said polypeptide.
 79. Acomposition comprising the polynucleotide of claim 68 and apharmaceutically acceptable carrier.